Methods for engineering t cells for immunotherapy by using rna-guided cas nuclease system

ABSTRACT

The present invention relates to methods of developing genetically engineered, preferably non-alloreactive T-cells for immunotherapy. This method involves the use of RNA-guided endonucleases, in particular Cas9/CRISPR system, to specifically target a selection of key genes in T-cells. The engineered T-cells are also intended to express chimeric antigen receptors (CAR) to redirect their immune activity towards malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies using T-Cells for treating cancer and viral infections.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 15, 2016, isnamed P81400433US00_SL.txt and is 47,236 bytes in size.

FIELD OF THE INVENTION

The present invention relates to methods of developing geneticallyengineered, preferably non-alloreactive T-cells for immunotherapy. Thismethod involves the use of RNA-guided endonucleases, in particularCas9/CRISPR system, to specifically target a selection of key geneticloci in T-cells. The engineered T-cells are also intended to expresschimeric antigen receptors (CAR) to redirect their immune activitytowards malignant or infected cells. The invention opens the way tostandard and affordable adoptive immunotherapy strategies using T-Cellsfor treating cancer, viral infections and auto-immune diseases.

BACKGROUND OF THE INVENTION

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific T cells generated ex vivo, is a promising strategy totreat viral infections and cancer. The T cells used for adoptiveimmunotherapy can be generated either by expansion of antigen-specific Tcells or redirection of T cells through genetic engineering (Park,Rosenberg et al. 2011). Transfer of viral antigen specific T cells is awell-established procedure used for the treatment of transplantassociated viral infections and rare viral-related malignancies.Similarly, isolation and transfer of tumor specific T cells has beenshown to be successful in treating melanoma.

Novel specificities in T cells have been successfully generated throughthe genetic transfer of transgenic T cell receptors or chimeric antigenreceptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptorsconsisting of a targeting moiety that is associated with one or moresignaling domains in a single fusion molecule. In general, the bindingmoiety of a CAR consists of an antigen-binding domain of a single-chainantibody (scFv), comprising the light and variable fragments of amonoclonal antibody joined by a flexible linker. Binding moieties basedon receptor or ligand domains have also been used successfully. Thesignaling domains for first generation CARs are derived from thecytoplasmic region of the CD3zeta or the Fc receptor gamma chains. Firstgeneration CARs have been shown to successfully redirect T cellcytotoxicity, however, they failed to provide prolonged expansion andanti-tumor activity in vivo. Signaling domains from co-stimulatorymolecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have beenadded alone (second generation) or in combination (third generation) toenhance survival and increase proliferation of CAR modified T cells.CARs have successfully allowed T cells to be redirected against antigensexpressed at the surface of tumor cells from various malignanciesincluding lymphomas and solid tumors (Jena, Dotti et al. 2010).

The current protocol for treatment of patients using adoptiveimmunotherapy is based on autologous cell transfer. In this approach, Tlymphocytes are recovered from patients, genetically modified orselected ex vivo, cultivated in vitro in order to amplify the number ofcells if necessary and finally infused into the patient. In addition tolymphocyte infusion, the host may be manipulated in other ways thatsupport the engraftment of the T cells or their participation in animmune response, for example pre-conditioning (with radiation orchemotherapy) and administration of lymphocyte growth factors (such asIL-2). Each patient receives an individually fabricated treatment, usingthe patient's own lymphocytes (i.e. an autologous therapy). Autologoustherapies face substantial technical and logistic hurdles to practicalapplication, their generation requires expensive dedicated facilitiesand expert personnel, they must be generated in a short time following apatient's diagnosis, and in many cases, pretreatment of the patient hasresulted in degraded immune function, such that the patient'slymphocytes may be poorly functional and present in very low numbers.Because of these hurdles, each patient's autologous cell preparation iseffectively a new product, resulting in substantial variations inefficacy and safety.

Ideally, one would like to use a standardized therapy in whichallogeneic therapeutic cells could be pre-manufactured, characterized indetail, and available for immediate administration to patients. Byallogeneic it is meant that the cells are obtained from individualsbelonging to the same species but are genetically dissimilar. However,the use of allogeneic cells presently has many drawbacks. Inimmune-competent hosts allogeneic cells are rapidly rejected, a processtermed host versus graft rejection (HvG), and this substantially limitsthe efficacy of the transferred cells. In immune-incompetent hosts,allogeneic cells are able to engraft, but their endogenous T-cellreceptors (TCR) specificities recognize the host tissue as foreign,resulting in graft versus host disease (GvHD), which can lead to serioustissue damage and death.

In order to effectively obtain allogeneic cells, the inventorspreviously disclosed a method to genetically engineer T-Cells, in whichdifferent effector genes, in particular those encoding T-cell receptors,were inactivated by using specific TAL-nucleases, better known under thetrade mark TALEN™ (Cellectis, 8, rue de la Croix Jarry, 75013 PARIS).This method has proven to be highly efficiency in primary cells usingRNA transfection as part of a platform allowing the mass production ofallogeneic T-cells (WO 2013/176915).

Recently, a new genome engineering tool has been developed based on thecomponents of the type II prokaryotic CRISPR (Clustered RegularlyInterspaced Short palindromic Repeats) adaptive immune system of thebacteria S. pyogenes. This multi-component system referred to asRNA-guided Cas nuclease system (Gasiunas, Barrangou et al. 2012; Jinek,Chylinski et al. 2012) or more simply as CRISPR, involves a Casendonuclease coupled with a guide RNA molecules that have the ability todrive said nuclease to some specific genome sequences. Where the RNAguide hybridizes the genome sequence, the endonuclease has the abilityto cleave the DNA. The CRISPR/CRISPR-associated (Cas) system involves 1)retention of foreign genetic material, called “spacers”, in clusteredarrays in the host genome, 2) expression of short guiding RNAs (crRNAs)from the spacers, 3) binding of the crRNAs to specific portions of theforeign DNA called protospacers and 4) degradation of protospacers byCRISPR-associated nucleases (Cas). The specificity of binding to theforeign DNA is controlled by the non-repetitive spacer elements in thepre-crRNA, which upon transcription along with the tracrRNA, directs theCas9 nuclease to the protospacer:crRNA heteroduplex and inducesdouble-strand breakage (DSB) formation. Additionally, the Cas9 nucleasecuts the DNA only if a specific sequence known as protospacer adjacentmotif (PAM) is present immediately downstream of the protospacersequence, whose canonical sequence in S. pyogenes is 5′-NGG-3′, where Nrefers to any nucleotide. Later on, it has been demonstrated that theexpression of a single chimeric crRNA:tracrRNA transcript, whichnormally is expressed as two different RNAs in the native type II CRISPRsystem, is sufficient to direct the Cas9 nuclease tosequence-specifically cleave target DNA sequences. By adapting theendogenous type II CRISPR/Cas system from S. pyogenes for use inmammalian cells, several groups have independently shown that RNA-guidedCas9 is able to efficiently introduce precise double stranded breaks atendogenous genomic loci in mammalian cells with high efficiencies andminimal off-target effects (Cong et al. 2013, Mali et al. 2013, Cho etal. 2013). In addition, several mutant forms of Cas9 nuclease have beendeveloped to take advantage of their features for additionalapplications in genome engineering and transcriptional regulation. Forinstance, one mutant form of Cas9 nuclease functions as a nickase (Jineket al. 2012), generating a break in complementary strand of DNA ratherthan both strands as with the wild-type Cas9. This allows repair of theDNA template using a high-fidelity pathway rather than NHEJ, whichprevents formation of indels at the targeted locus, and possibly otherlocations in the genome to reduce possible off-target/toxicity effectswhile maintaining ability to undergo homologous recombination (Cong etal., 2013). Most recently, paired nicking has been shown to reduceoff-target activity by 50- to 1,500 fold in cell lines and to facilitategene knockout in mouse zygote without losing on-target cleavageefficiency (Ran et al., 2013).

Although RNA-guided endonucleases, such as the Cas9/CRISPR systemappears to be an attractive approach for genetically engineeringmammalian cells, the use thereof in primary cells, in particular inT-Cells, is significantly hurdled by the fact that:

-   -   T-cells are adversely affected by the introduction of DNA in        their cytoplasm: high rate of apoptosis is observed when        transforming cells with DNA vectors;    -   CRISPR system requires stable expression of Cas9 in the cells.        However, prolonged expression of Cas9 in living cells may lead        to chromosomal defects;    -   Specificity of current RNA-guided endonuclease is determined        only by sequences comprising 11 nucleotides (N12-20NGG, where        NGG represents the PAM), which makes it very difficult to        identify target sequences in desired loci that are unique in the        genome.

The present application aims to provide solutions to these limitationsin order to efficiently implement RNA-guided endonucleases into T-cells.

SUMMARY OF THE INVENTION

The present invention discloses methods to engineer T cells, inparticular allogeneic T cells obtainable from donors, to make themsuitable for immunotherapy purposes, by using RNA-guided endonucleasessuch as Cas9/CRISPR.

The methods of the present invention more particularly allow the precisemodification of the genome of T-cells relevant for immunotherapy byinactivating or replacing genes involved in MHC recognition and/orimmune checkpoint proteins. In particular, they provide specificrelevant targets sequences in the genome for the guide RNA to inactivatecomponents of TCR without provoking death of the cells.

According to several preferred embodiments, the modified cells relevantfor immunotherapy further comprise exogenous recombinant polynucleotidesencoding single-chain and multi-subunit chimeric antigen receptor (CARs)for specific cell recognition. More particularly, modified T-cells fortreating lymphoma are made available bearing CAR directed against CD19antigen.

The present invention encompasses the isolated cells or cell linescomprising the genetic modifications set forth in the detaileddescription, examples and figures, as well as any of the proteins,polypeptides or vectors useful to implement RNA-guided endonucleases inT-cells.

As a result of the invention, modified T-cells can be used astherapeutic products, ideally as an “off the shelf” product, in methodsfor treating or preventing cancer, infections or auto-immune disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the normal relationship betweenT-cells and antigen presenting cell.

FIG. 2: Schematic representation of the genetically modified therapeuticT-cells according to the invention against the patient's tumor cells.

FIG. 3: Schematic representation of a multi-subunit CAR, which canintroduced into the RNA guided endonuclease engineered T-cells accordingto the invention.

FIG. 4: Schematic representation of one example of the method ofengineering human allogenic cells for immunotherapy.

FIG. 5: Flow cytometry analysis of the activity of CRISPR system in Tcells with respect to TCR gene (experimental results from example2)—5×10⁶ T cells were transfected with 10 μg Cas9 mRNA+10 μg plasmid DNAexpressing sgRNA specific for TRAC genes under the control of U6promoter in the presence of a caspase inhibitor. Readout: 3 days posttransfection, flow cytometry analysis using TCRαβ antibody. Controls:untransfected cells.

FIG. 6: Flow cytometry analysis of the activity of CRISPR system in Tcells with respect to CD52 gene (experimental results from example2)—5×10⁶ T cells were transfected with 10 μg Cas9 mRNA+10 μg plasmid DNAexpressing sgRNA specific for CD52 genes under the control of U6promoter in the presence of a caspase inhibitor. Readout: 3 days posttransfection, flow cytometry analysis using CD52 antibody. Controls:untransfected cells.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific termsused have the same meaning as commonly understood by a skilled artisanin the fields of gene therapy, biochemistry, genetics, and molecularbiology.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willprevail. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, CurrentProtocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andson Inc, Library of Congress, USA); Molecular Cloning: A LaboratoryManual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J.Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Harries & S. J. Higgins eds. 1984); TranscriptionAnd Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture OfAnimal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); ImmobilizedCells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelsonand M. Simon, eds.-in-chief, Academic Press, Inc., New York),specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “GeneExpression Technology” (D. Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

In a general aspect, the present invention relates to methods for newadoptive immunotherapy strategies in treating cancer and infections.

As a main objective of the invention is the use of RNA-guidedendonuclease, such as Cas9 to genetically modify T-cells for producingcells suitable in cell therapy.

In a first embodiment, the method of the invention concerns a method ofpreparing T-cells for immunotherapy comprising the step of:

-   -   (a) Genetically modifying T-cells by introduction and/or        expression into the cells of at least:        -   a RNA-guided endonuclease; and        -   a specific guide RNA that directs said endonuclease to at            least one targeted locus in the T-cell genome    -   (b) expanding the resulting cells.

By “RNA-guided endonuclease” is meant a polypeptide which endonucleaseactivity and specificity depend on its association with a RNA molecule.The full sequence of this RNA molecule or more generally a fragment ofthis RNA molecule, which is a sequence preferably longer than 8 nucleicacid bases, more preferably longer than 10 nucleic acid bases, even morepreferably longer than 12 nucleic acid bases, has the ability to specifya target sequence in the genome. In general, this RNA molecule has theability to hybridize said target sequence and to mediate theendonuclease activity of said endonuclease. An example of RNA-guidedendonuclease is cas9 as part of the Cas9/CRISPR system.

Cas 9

Cas9, also named Csn1 (COG3513) is a large protein that participates inboth crRNA biogenesis and in the destruction of invading DNA. Cas9 hasbeen described in different bacterial species such as S. thermophilus(Sapranauskas, Gasiunas et al. 2011), Listeria innocua (Gasiunas,Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and S. pyogenes(Deltcheva, Chylinski et al. 2011). The large Cas9 protein (>1200 aminoacids) contains two predicted nuclease domains, namely HNH (McrA-like)nuclease domain that is located in the middle of the protein and asplitted RuvC-like nuclease domain (RNase H fold) (Haft, Selengut et al.2005; Makarova, Grishin et al. 2006).

By “Cas9” is also meant an engineered endonuclease or a homologue ofCas9 which is capable of processing target nucleic acid sequence. Inparticular embodiment, Cas9 can induce a cleavage in the nucleic acidtarget sequence which can correspond to either a double-stranded breakor a single-stranded break. Cas9 variant can be a Cas9 endonuclease thatdoes not naturally exist in nature and that is obtained by proteinengineering or by random mutagenesis. Cas9 variants according to theinvention can for example be obtained by mutations i.e. deletions from,or insertions or substitutions of at least one residue in the amino acidsequence of a S. pyogenes Cas9 endonuclease (COG3513—SEQ ID NO. 3). Inthe frame aspects of the present invention, such Cas9 variants remainfunctional, i.e. they retain the capacity of processing a target nucleicacid sequence. Cas9 variant can also be homologues of S. pyogenes Cas9which can comprise deletions from, or insertions or substitutions of, atleast one residue within the amino acid sequence of S. pyogenes Cas9.Any combination of deletion, insertion, and substitution may also bemade to arrive at the final construct, provided that the final constructpossesses the desired activity, in particular the capacity of binding aguide RNA or nucleic acid target sequence.

RuvC/RNaseH motif includes proteins that show wide spectra ofnucleolytic functions, acting both on RNA and DNA (RNaseH, RuvC, DNAtransposases and retroviral integrases and PIWI domain of Argonautproteins). In the present invention the RuvC catalytic domain of theCas9 (SEQ ID NO. 4) protein can be characterized by the sequence motif:D-[I/L]-G-X-X-S-X-G-W-A, wherein X represents any one of the natural 20amino acids and [I/L] represents isoleucine or leucine (SEQ ID NO: 1).In other terms, the present invention relates to Cas9 variant whichcomprises at least D-[I/L]-G-X-X-S-X-G-W-A sequence, wherein Xrepresents any one of the natural 20 amino acids and [I/L] representsisoleucine or leucine (SEQ ID NO: 1).

HNH motif is characteristic of many nucleases that act ondouble-stranded DNA including colicins, restriction enzymes and homingendonucleases. The domain HNH (SMART ID: SM00507, SCOP nomenclature:HNHfamily) is associated with a range of DNA binding proteins, performing avariety of binding and cutting functions (Gorbalenya 1994; Shub,Goodrich-Blair et al. 1994). Several of the proteins are hypothetical orputative proteins of no well-defined function. The ones with knownfunction are involved in a range of cellular processes includingbacterial toxicity, homing functions in groups I and II introns andinteins, recombination, developmentally controlled DNA rearrangement,phage packaging, and restriction endonuclease activity (Dalgaard, Klaret al. 1997). These proteins are found in viruses, archaebacteria,eubacteria, and eukaryotes. Interestingly, as with the LAGLI-DADG (SEQID NO: 58) and the GIY-YIG motifs, the HNH motif is often associatedwith endonuclease domains of self-propagating elements like inteins,Group I, and Group II introns (Gorbalenya 1994; Dalgaard, Klar et al.1997). The HNH domain can be characterized by the presence of aconserved Asp/His residue flanked by conserved His (amino-terminal) andHis/Asp/Glu (carboxy-terminal) residues at some distance. A substantialnumber of these proteins can also have a CX2C motif on either side ofthe central Asp/His residue. Structurally, the HNH motif appears as acentral hairpin of twisted β-strands, which are flanked on each side byan a helix (Kleanthous, Kuhlmann et al. 1999). The large HNH domain ofCas9 is represented by SEQ ID NO. 5. In the present invention, the HNHmotif can be characterized by the sequence motif:Y-X-X-D-H-X-X-P-X-S-X-X-X-D-X-S, wherein X represents any one of thenatural 20 amino acids (SEQ ID NO: 2). The present invention relates toa Cas9 variant which comprises at least Y-X-X-D-H-X-X-P-X-S-X-X-X-D-X-Ssequence wherein X represents any one of the natural 20 amino acids (SEQID NO: 2).

This invention can be of particular interest to easily do targetedmultiplex gene modifications and to create an inducible nuclease systemby introduction of the guide RNA to the Cas9 cells. For the purpose ofthe present invention, the inventors have established that Cas9 proteincan be divided into two separate split Cas9 RuvC and HNH domains whichcan process target nucleic acid sequence together or separately with theguide RNA.

Also the RuvC and HNH domains from different RNA guided endonucleases orCas homologues may be assembled to improve nuclease efficiency orspecificity. The domains from different species can be either split intotwo proteins or fused to each other to form a variant Cas protein. TheCas9 split system is deemed particularly suitable for an induciblemethod of genome targeting and to avoid the potential toxic effect ofthe Cas9 overexpression within the cell. Indeed, a first split Cas9domain can be introduced into the cell, preferably by stablytransforming said cell with a transgene encoding said split domain.Then, the complementary split part of Cas9 can be introduced into thecell, such that the two split parts reassemble into the cell toreconstitute a functional Cas9 protein at the desired time.

The reduction of the size of the split Cas9 compared to wild type Cas9ease the vectorization and the delivery into the cell, for example, byusing cell penetrating peptides. Re-arranging domains from different Casproteins, allows to modulate the specificity and nuclease activity, forinstance, by targeting PAM motifs that are slightly different from S.pyogenes Cas9

Split Cas9 System

The previous characterization of the RuvC and HNH domains has promptedthe inventors to engineer Cas9 protein to create split Cas9 protein.Surprisingly, the inventors showed that these two split Cas9 couldprocess together or separately the nucleic acid target. This observationallows developing a new Cas9 system using split Cas9 protein. Each splitCas9 domains can be prepared and used separately. Thus, this splitsystem displays several advantages for vectorization and delivery of theRNA guided endonuclease in T-cells, allowing delivering a shorter and/orinactive protein, and is particularly suitable to induce genomeengineering in T-cells at the desired time and thus limiting thepotential toxicity of an integrated Cas9 nuclease.

By “Split Cas9” is meant here a reduced or truncated form of a Cas9protein or Cas9 variant, which comprises either a RuvC or HNH domain,but not both of these domains. Such “Split Cas9” can be usedindependently with guide RNA or in a complementary fashion, like forinstance, one Split Cas9 providing a RuvC domain and another providingthe HNH domain. Different split RNA guided endonucleases may be usedtogether having either RuvC and/or NHN domains.

Each Cas9 split domain can be derived from the same or from differentCas9 homologues. Many homologues of Cas9 have been identified in genomedatabases.

As a method of genome engineering the invention provides with the stepsof:

-   -   (a) selecting a target nucleic acid sequence, optionally        comprising a PAM motif in the cell;    -   (b) providing a guide RNA comprising a sequence complementary to        the target nucleic acid sequence;    -   (c) providing at least one split Cas9 domain;    -   (d) introducing into the cell the guide RNA and said split Cas9        domain(s), such that split Cas9 domain(s) processes the target        nucleic acid sequence in the cell.

Said Cas9 split domains (RuvC and HNH domains) can be simultaneously orsequentially introduced into the cell such that said split Cas9domain(s) process the target nucleic acid sequence in the cell. SaidCas9 split domains and guide RNA can be introduced into the cell byusing cell penetrating peptides or other transfection methods asdescribed below.

In another aspect of the invention, only one split Cas9 domain, referredto as compact Cas9 is introduced into said cell. Indeed, surprisinglythe inventors showed that the split Cas9 domain comprising the RuvCmotif as described above is capable of cleaving a target nucleic acidsequence independently of split domain comprising the HNH motif. Thus,they could establish that the guideRNA does not need the presence of theHNH domain to bind to the target nucleic acid sequence and issufficiently stable to be bound by the RuvC split domain. In a preferredembodiment, said split Cas9 domain alone is capable of nicking saidtarget nucleic acid sequence.

Each split domain can be fused to at least one active domain in theN-terminal and/or C-terminal end, said active domain can be selectedfrom the group consisting of: nuclease (e.g. endonuclease orexonuclease), polymerase, kinase, phosphatase, methylase, demethylase,acetylase, desacetylase, topoisomerase, integrase, transposase, ligase,helicase, recombinase, transcriptional activator (e.g. VP64, VP16),transcriptional inhibitor (e. g; KRAB), DNA end processing enzyme (e.g.Trex2, Tdt), reporter molecule (e.g. fluorescent proteins, lacZ,luciferase).

HNH domain is responsible for nicking of one strand of the targetdouble-stranded DNA and the RuvC-like RNaseH fold domain is involved innicking of the other strand (comprising the PAM motif) of thedouble-stranded nucleic acid target (Jinek, Chylinski et al. 2012).However, in wild-type Cas9, these two domains result in blunt cleavageof the invasive DNA within the same target sequence (proto-spacer) inthe immediate vicinity of the PAM (Jinek, Chylinski et al. 2012). Cas 9can be a nickase and induces a nick event within different targetsequences.

As non-limiting example, Cas9 or split Cas9 can comprise mutation(s) inthe catalytic residues of either the HNH or RuvC-like domains, to inducea nick event within different target sequences. As non-limiting example,the catalytic residues of the Cas9 protein are those corresponding toamino acids D10, D31, H840, H868, N882 and N891 or aligned positionsusing CLUSTALW method on homologues of Cas Family members. Any of theseresidues can be replaced by any other amino acids, preferably by alanineresidue. Mutation in the catalytic residues means either substitution byanother amino acids, or deletion or addition of amino acids that inducethe inactivation of at least one of the catalytic domain of cas9. (cf.In a particular embodiment, Cas9 or split Cas9 may comprise one orseveral of the above mutations. In another particular embodiment, splitCas9 comprises only one of the two RuvC and HNH catalytic domains. Inthe present invention, Cas9 from different species, Cas9 homologues,Cas9 engineered and functional variant thereof can be used. Theinvention envisions the use of any RNA guided endonuclease or split RNAguided endonucleases variants to perform nucleic acid cleavage in agenetic sequence of interest.

Preferably, said Cas9 variants have an amino acid sequence sharing atleast 70%, preferably at least 80%, more preferably at least 90%, andeven more preferably 95% identity with Cas9 of S. pyogenes (COG3513—SEQID NO. 3).

In another aspect of the present invention, Cas9 or split Cas9 lack ofendonucleolytic activity. The resulting Cas9 or split Cas9 isco-expressed with guide RNA designed to comprises a complementarysequence of the target nucleic acid sequence. Expression of Cas9 lackingendonucleolytic activity yields to specific silencing of the gene ofinterest. This system is named CRISPR interference (CRISPRi) (Qi, Larsonet al. 2013). By silencing, it is meant that the gene of interest is notexpressed in a functional protein form. The silencing may occur at thetranscriptional or the translational step. According to the presentinvention, the silencing may occur by directly blocking transcription,more particularly by blocking transcription elongation or by targetingkey cis-acting motifs within any promoter, sterically blocking theassociation of their cognate trans-acting transcription factors. TheCas9 lacking endonucleolytic activity comprises both non-functional HNHand RuvC domains. In particular, the Cas9 or split Cas9 polypeptidecomprises inactivating mutations in the catalytic residues of both theRuvC-like and HNH domains. For example, the catalytic residues requiredfor cleavage Cas9 activity can be D10, D31, H840, H865, H868, N882 andN891 of Cas9 of S. pyogenes (COG3513—SEQ ID NO. 3) or aligned positionsusing CLUSTALW method on homologues of Cas Family members. The residuescomprised in HNH or RuvC motifs can be those described in the aboveparagraph. Any of these residues can be replaced by any one of the otheramino acids, preferably by alanine residue. Mutation in the catalyticresidues means either substitution by another amino acids, or deletionor addition of amino acids that induce the inactivation of at least oneof the catalytic domain of cas9.

In another particular embodiment, Cas9 or each split domains can befused to at least one active domain in the N-terminal and/or C-terminalend. Said active domain can be selected from the group consisting of:nuclease (e.g. endonuclease or exonuclease), polymerase, kinase,phosphatase, methylase, demethylase, acetylase, desacetylase,topoisomerase, integrase, transposase, ligase, helicase, recombinase,transcriptional activator (e.g. VP64, VP16), transcriptional inhibitor(e. g; KRAB), DNA end processing enzyme (e.g. Trex2, Tdt), reportermolecule (e.g. fluorescent proteins, lacZ, luciferase).

Inducible Nuclease Activity for the RNA Guided Endonuclease/Guided RNASystem

Given the potential toxicity of the RNA guided endonuclease within theT-cells, due to possible unspecific interactions with various RNAs inthe cell and expectable off-site targeting, the inventors have soughtfor solutions to induce the nuclease activity of the RNA-guidedendonuclease transiently, ideally during the life-span of the guide RNAinto the cells.

As a primary solution, the RNA-guided endonuclease can be expressedunder a stabilized or inactive form, which is made active uponactivation by an enzyme produced by the T-cell or destabilization of itspolypeptide structure inside the T-cell. Conditional protein stabilitycan be obtained for instance by fusion of the endonuclease to astabilizing/destabilizing protein based, as a non-limiting example, onthe FKBP/rapamycin system, where protein conformational change inducedby a small molecule.

Chemical or light induced dimerization of a protein partner fused to theendonuclease protein can also be used to lock or unlock theendonuclease.

In the situation where the RNA guided endonuclease is split in twopolypeptides as suggested before, each split can be fused to a partnerprotein. Both partner proteins will dimerize upon addition of a smallmolecule and reconstitute, in living cells, an active endonuclease. Suchsystems can be based, as non-limiting example, on the use of FKBB/FRB asdimerization partners and rapamycin as a small molecule. As anotherexample, protein that can undergo a major conformational change uponbinding to a small molecule or metabolite inserted in the endonucleaseprotein (1 polypeptide chain composed of 2 “splits”). Binding (or not)of the small molecule will switch the Cas9 between an active andinactive conformation. Such systems can be based, as non-limitingexample, on the use of calmodulin and Ca2+.

Each half of the split endonuclease can also be fused to a partnerprotein sensitive to light. Such systems can rely, as non-limitingexample, on blue light with the Cryptochrome 2 (CRY2) and CIB1 as fusionpartners or on ultraviolet light with the ultraviolet-B photoreceptorUVR8 and COP1 fusion partners. Both light and chemical may also becombined by using, for instance, the Phytochrome B and PIF6 partners(red light association, far red light dissociation) and an exogenous PCBchromophore.

Guide RNA

The method of the present invention comprises providing an engineeredguide RNA. Guide RNA corresponds to a nucleic acid sequence comprising acomplementary sequence. Preferably, said guide RNA correspond to a crRNAand tracrRNA which can be used separately or fused together.

In natural type II CRISPR system, the CRISPR targeting RNA (crRNA)targeting sequences are transcribed from DNA sequences known asprotospacers. Protospacers are clustered in the bacterial genome in agroup called a CRISPR array. The protospacers are short sequences (˜20bp) of known foreign DNA separated by a short palindromic repeat andkept like a record against future encounters. To create the crRNA, theCRISPR array is transcribed and the RNA is processed to separate theindividual recognition sequences between the repeats. Thespacer-containing CRISPR locus is transcribed in a long pre-crRNA. Theprocessing of the CRISPR array transcript (pre-crRNA) into individualcrRNAs is dependent on the presence of a trans-activating crRNA(tracrRNA) that has sequence complementary to the palindromic repeat.The tracrRNA hybridizes to the repeat regions separating the spacers ofthe pre-crRNA, initiating dsRNA cleavage by endogenous RNase III, whichis followed by a second cleavage event within each spacer by Cas9,producing mature crRNAs that remain associated with the tracrRNA andCas9 and form the Cas9-tracrRNA:crRNA complex. Engineered crRNA withtracrRNA is capable of targeting a selected nucleic acid sequence,obviating the need of RNase III and the crRNA processing in general(Jinek, Chylinski et al. 2012).

In the present invention, crRNA is engineered to comprise a sequencecomplementary to a portion of a target nucleic acid such that it iscapable of targeting, preferably cleaving the target nucleic acidsequence. In a particular embodiment, the crRNA comprises a sequence of5 to 50 nucleotides, preferably 12 nucleotides which is complementary tothe target nucleic acid sequence. In a more particular embodiment, thecrRNA is a sequence of at least 30 nucleotides which comprises at least10 nucleotides, preferably 12 nucleotides complementary to the targetnucleic acid sequence.

In another aspect, crRNA can be engineered to comprise a larger sequencecomplementary to a target nucleic acid. Indeed, the inventors showedthat the RuvC split Cas9 domain is able to cleave the target nucleicacid sequence only with a guide RNA. Thus, the guide RNA can bind thetarget nucleic acid sequence in absence of the HNH split Cas9 domain.The crRNA can be designed to comprise a larger complementary sequence,preferably more than 20 bp, to increase the annealing between DNA-RNAduplex without the need to have the stability effect of the HNH splitdomain binding. Thus, the crRNA can comprise a complementary sequence toa target nucleic acid sequence of more than 20 bp. Such crRNA allowincreasing the specificity of the Cas9 activity.

The crRNA may also comprise a complementary sequence followed by 4-10nucleotides on the 5′end to improve the efficiency of targeting (Cong,Ran et al. 2013; Mali, Yang et al. 2013). In preferred embodiment, thecomplementary sequence of the crRNA is followed in 3′end by a nucleicacid sequence named repeat sequences or 3′extension sequence.

Co-expression of several crRNA with distinct complementary regions totwo different genes targeted both genes can be used simultaneously.Thus, in particular embodiment, the crRNA can be engineered to recognizedifferent target nucleic acid sequences simultaneously.

In this case, same crRNA comprises at least two distinct sequencescomplementary to a portion of the different target nucleic acidsequences. In a preferred embodiment, said complementary sequences arespaced by a repeat sequence.

As a further embodiment of the present invention, said guide RNA is bornby a peptide nucleic acid (PNA) or Locked Nucleic Acid (LNA), inparticular to improve stability of said guide RNA in the T-cells.

PAM Motif

Any potential selected target nucleic acid sequence in the presentinvention may have a specific sequence on its 3′ end, named theprotospacer adjacent motif or protospacer associated motif (PAM). ThePAM is present in the targeted nucleic acid sequence but not in thecrRNA that is produced to target it. Preferably, the proto-spaceradjacent motif (PAM) may correspond to 2 to 5 nucleotides startingimmediately or in the vicinity of the proto-spacer at the leader distalend. The sequence and the location of the PAM vary among the differentsystems. PAM motif can be for examples NNAGAA, NAG, NGG, NGGNG, AWG, CC,CC, CCN, TCN, TTC as non-limiting examples (Shah S A, RNA biology 2013).Different Type II systems have differing PAM requirements. For example,the S. pyogenes system requires an NGG sequence, where N can be anynucleotides. S. thermophilus Type II systems require NGGNG (Horvath andBarrangou 2010) and NNAGAAW (Deveau, Barrangou et al. 2008), whiledifferent S. mutant systems tolerate NGG or NAAR (Van der Ploeg 2009).PAM is not restricted to the region adjacent to the proto-spacer but canalso be part of the proto-spacer (Mojica, Diez-Villasenor et al. 2009).In a particular embodiment, the Cas9 protein can be engineered not torecognize any PAM motif or to recognize a non-natural PAM motif. In thiscase, the selected target sequence may comprise a smaller or a largerPAM motif with any combinations of amino acids. In a preferredembodiment, the selected target sequence comprise a PAM motif whichcomprises at least 3, preferably, 4, more preferably 5 nucleotidesrecognized by the Cas9 variant according to the present invention.

Coexpression of several crRNA with distinct complementary regions to twodifferent genes targeted both genes can be used simultaneously. Thus, inparticular embodiment, the crRNA can be engineered to recognizedifferent target nucleic acid sequences simultaneously. In this case,same crRNA comprises at least two distinct sequences complementary to aportion of the different target nucleic acid sequences. In a preferredembodiment, said complementary sequences are spaced by a repeatsequence.

The crRNA according to the present invention can also be modified toincrease its stability of the secondary structure and/or its bindingaffinity for Cas9. In a particular embodiment, the crRNA can comprise a2′, 3′-cyclic phosphate. The 2′, 3′-cyclic phosphate terminus seems tobe involved in many cellular processes i.e. tRNA splicing,endonucleolytic cleavage by several ribonucleases, in self-cleavage byRNA ribozyme and in response to various cellular stress includingaccumulation of unfolded protein in the endoplasmatic reticulum andoxidative stress (Schutz, Hesselberth et al. 2010). The inventors havespeculated that the 2′, 3′-cyclic phosphate enhances the crRNA stabilityor its affinity/specificity for Cas9. Thus, the present inventionrelates to the modified crRNA comprising a 2′, 3′-cyclic phosphate, andthe methods for genome engineering based on the CRISPR/cas system(Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al.2013) using the modified crRNA.

The guide RNA may also comprise a Trans-activating CRISPR RNA(TracrRNA). Trans-activating CRISPR RNA according to the presentinvention are characterized by an anti-repeat sequence capable ofbase-pairing with at least a part of the 3′ extension sequence of crRNAto form a tracrRNA:crRNA also named guide RNA (gRNA). TracrRNA comprisesa sequence complementary to a region of the crRNA. A guide RNAcomprising a fusion of crRNA and tracrRNA that forms a hairpin thatmimics the tracrRNA-crRNA complex (Jinek, Chylinski et al. 2012; Cong,Ran et al. 2013; Mali, Yang et al. 2013) can be used to direct Cas9endonuclease-mediated cleavage of target nucleic acid. The guide RNA maycomprise two distinct sequences complementary to a portion of the twotarget nucleic acid sequences, preferably spaced by a repeat sequence.

Homologous Recombination

Endonucleolytic breaks are known to stimulate the rate of homologousrecombination. Therefore, as another preferred embodiment, the presentinvention relates to a method for inducing homologous gene targeting inthe nucleic acid target sequence by using a RNA guided endonuclease.This method can further comprise the step of providing an exogeneousnucleic acid to the cell (e.g. donor DNA) comprising at least a sequencehomologous to a portion of the target nucleic acid sequence, such thathomologous recombination occurs between the target nucleic acid sequenceand said exogeneous nucleic acid.

In particular embodiments, said exogenous nucleic acid comprises firstand second portions which are homologous to region 5′ and 3′ of thetarget nucleic acid sequence, respectively. Said exogenous nucleic acidin these embodiments also comprises a third portion positioned betweenthe first and the second portion which comprises no homology with theregions 5′ and 3′ of the target nucleic acid sequence. Followingcleavage of the target nucleic acid sequence, a homologous recombinationevent is stimulated between the target nucleic acid sequence and theexogenous nucleic acid. Preferably, homologous sequences of at least 50bp, preferably more than 100 bp and more preferably more than 200 bp areused within said donor matrix. Therefore, the homologous sequence ispreferably from 200 bp to 6000 bp, more preferably from 1000 bp to 2000bp. Indeed, shared nucleic acid homologies are located in regionsflanking upstream and downstream the site of the break and the nucleicacid sequence to be introduced should be located between the two arms.

Depending on the location of the target nucleic acid sequence whereinbreak event has occurred, such exogenous nucleic acid can be used toknock-out a gene, e.g. when exogenous nucleic acid is located within theopen reading frame of said gene, or to introduce new sequences or genesof interest. Sequence insertions by using such exogenous nucleic acidcan be used to modify a targeted existing gene, by correction orreplacement of said gene (allele swap as a non-limiting example), or toup- or down-regulate the expression of the targeted gene (promoter swapas non-limiting example), said targeted gene correction or replacement.

Delivery Methods

The methods of the invention involve introducing molecule of interestsuch as guide RNA (crRNA, tracrRNa, or fusion guide RNA), split Cas9,Cas9, exogenous nucleic acid, DNA end-processing enzyme into a cell.Guide RNA, split Cas9, Cas9, exogenous nucleic acid, DNA end-processingenzyme or others molecules of interest may be synthesized in situ in thecell as a result of the introduction of polynucleotides, preferablytransgenes, comprised in vector encoding RNA or polypeptides into thecell. Alternatively, the molecule of interest could be produced outsidethe cell and then introduced thereto.

The inventors have considered any means known in the art to allowdelivery inside cells or subcellular compartments of agents/chemicalsand molecules (proteins and nucleic acids) can be used includingliposomal delivery means, polymeric carriers, chemical carriers,lipoplexes, polyplexes, dendrimers, nanoparticles, emulsion, naturalendocytosis or phagocytose pathway as non-limiting examples, as well asphysical methods such as electroporation.

As a preferred embodiment of the invention, polynucleotides encoding theRNA guided endonuclease of the present invention are transfected undermRNA form, which is introduced directly into the cells, for example byelectroporation. The inventors have determined different optimalconditions for mRNA electroporation in T-cell displayed in Table 1. Theinventor used the cytoPulse technology which allows, by the use ofpulsed electric fields, to transiently permeabilize living cells fordelivery of material into the cells. The technology, based on the use ofPulseAgile (Harvard Apparatus, Holliston, Mass. 01746 USA)electroporation waveforms grants the precise control of pulse duration,intensity as well as the interval between pulses (U.S. Pat. No.6,010,613 and WO2004083379). All these parameters can be modified inorder to reach the best conditions for high transfection efficiency withminimal mortality. Basically, the first high electric field pulses allowpore formation, while subsequent lower electric field pulses allow tomove the polynucleotide into the cell. In one aspect of the presentinvention, the inventor describe the steps that led to achievementof >95% transfection efficiency of mRNA in T cells, and the use of theelectroporation protocol to transiently express different kind ofproteins in T cells. In particular the invention relates to a method oftransforming T cell comprising contacting said T cell with RNA andapplying to T cell an agile pulse sequence consisting of:

-   -   (a) one electrical pulse with a voltage range from 2250 to 3000        V per centimeter, a pulse width of 0.1 ms and a pulse interval        of 0.2 to 10 ms between the electrical pulses of step (a) and        (b);    -   (b) one electrical pulse with a voltage range from 2250 to 3000        V with a pulse width of 100 ms and a pulse interval of 100 ms        between the electrical pulse of step (b) and the first        electrical pulse of step (c); and    -   (c) 4 electrical pulses with a voltage of 325 V with a pulse        width of 0.2 ms and a pulse interval of 2 ms between each of 4        electrical pulses.        In particular embodiment, the method of transforming T cell        comprising contacting said T cell with RNA and applying to T        cell an agile pulse sequence consisting of:    -   (a) one electrical pulse with a voltage of 2250, 2300, 2350,        2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900        or 3000V per centimeter, a pulse width of 0.1 ms and a pulse        interval of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ms between        the electrical pulses of step (a) and (b);    -   (b) one electrical pulse with a voltage range from 2250, of        2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500,        2600, 2700, 2800, 2900 or 3000V with a pulse width of 100 ms and        a pulse interval of 100 ms between the electrical pulse of        step (b) and the first electrical pulse of step (c); and    -   (c) 4 electrical pulses with a voltage of 325 V with a pulse        width of 0.2 ms and a pulse interval of 2 ms between each of 4        electrical pulses.        Any values included in the value range described above are        disclosed in the present application. Electroporation medium can        be any suitable medium known in the art. Preferably, the        electroporation medium has conductivity in a range spanning 0.01        to 1.0 milliSiemens.

TABLE 1 Different cytopulse programs used to determine the minimalvoltage required for electroporation in PBMC derived T-cells. Cyto-Group 1 Group 2 Group 3 pulse duration Interval duration Intervalduration Interval program Pulses V (ms) (ms) Pulses V (ms) (ms) Pulses V(ms) (ms) 1 1 600 0.1 0.2 1 600 0.1 100 4 130 0.2 2 2 1 900 0.1 0.2 1900 0.1 100 4 130 0.2 2 3 1 1200 0.1 0.2 1 1200 0.1 100 4 130 0.2 2 4 11200 0.1 10 1 900 0.1 100 4 130 0.2 2 5 1 900 0.1 20 1 600 0.1 100 4 1300.2 2

Viral Transduction

According to the present invention, the use of viral vectors as definedhereafter for transduction of the nucleic acids encoding the RNA-guidedendonucleases and/or the transcripts to be used as guide RNA was foundto be a possible alternative to the previous means of transfection.Methods for viral transduction are well known in the art. However, suchmethods may encounter some limitations in the present situation due tothe stable integration of the retroviral or lentiviral vectors in theT-cells genome and the resulting expression of the endonuclease over along period of time. This may have deleterious effects on the T-cellsdepending on the level of specificity conferred by the RNA guide.

Pseudo-Transduction Using Viral Encapsidation of RNA-Guided Endonuclease

Given some of the disadvantages of existing means and materials todeliver RNA-guided endonucleases to their sites of action in T-cells,alternative delivery methods have been sought by the inventors, inparticular delivery methods where the RNA-guided endonucleases isintroduced under polypeptide form by fusing said polypeptide viralcomponents. As an example, the RNA-guided endonucleases can be fused toproteins of the HIV pre-integration complex, such as Vpr or Vpx.Accordingly, RNA-guided endonucleases under polypeptide form can actupon a specific target in the host cell genome as soon as they arereleased into the cell following virus entry and decapsulation.

For therapeutic application, where the use of the above retroviralcomponents may raise issues, because they are “helper proteins” foropportunistic viral infections, an alternative could be unexpectedlyfound by the inventors. They have observed that RNA-guidedendonucleases, in particular Cas9 could be incorporated into lentiviralvector particles during virion assembly and that such ‘self’incorporated endonucleases are sufficient to act so as to modify atarget genome following infection of a target cell by such a virusvector.

In accordance with this aspect of the invention, there is provided amethod of genome engineering involving one or several of the steps of:

a) assembling at least one virus vector in the presence of anendonuclease; said endonuclease being preferably not fused with anylentiviral component

b) bringing at least one target cell into contact with said virusvector,

c) wherein following internalization of said virus vector by said targetcell, said nuclease recognizes and cleaves at least one specific targetin the genome of said at least one target cell.

In accordance with the present invention viral vectors include thosederived from a virus such as a retrovirus, adenovirus, parvovirus (e. g.adenoassociated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabiesand vesicular stomatitis virus), paramyxovirus (e. g. measles andSendai), positive strand RNA viruses such as picornavirus andalphavirus, and double-stranded DNA viruses including adenovirus,herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include: avian leukosis-sarcoma, mammalianC-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, In Fundamental Virology, Third Edition, B. N. Fields, etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). The terms“viral vector” or “vector” refers to a modified virus particle which canbe used to introduce a nucleic acid molecule and/or a peptide or othermolecule into a target cell.

The inventors have shown that following transfer into the cellcytoplasm, the RNA-guided endonuclease retains its activity and canreadily associate with the guide RNA. Said guide RNA can also beencapsidated during virion assembly and thereby simultaneously releasedin the T-cells cytoplasm.

In accordance with a further aspect of the present invention the virusvector is a Lentiviral or retroviral based vector (LV), which arepreferably non-integrative. LVs are replication defective viralparticles which comprise an inner protein core surrounding the geneticmaterial of the virus, generally called the nucleocapsid core and anouter lipid membrane. These replication defective viral particles areassembled by expressing proteins encoded by the lentiviral gag and polgenes in packaging cells. The gag and pol genes encode polyproteins anda protease that processes these polyproteins into individual componentsof the virus particle.

A NLS, an amino acid sequence which acts to drive the protein to thecell nucleus through the Nuclear Pore Complex can be fused to the RNAguided endonuclease in order to improve its delivery efficiency.Typically, a NLS consists of one or more short sequences of positivelycharged amino acids such as lysines or arginines such as those from theknown proteins SV40 large T antigen—PKKKRKV—(SEQ ID NO: 59),nucleoplasmin—KR[PAATKKAGQA]KKKK—(SEQ ID NO: 60), p54—RIRKKLR—(SEQ IDNO: 61), SOX9—PRRRK—(SEQ ID NO: 62), NS5A—PPRKKRTVV—(SEQ ID NO: 63).

Independently from the present use of RNA-guided endonucleases accordingto the invention, the above method of viral encapsidation can beexpanded to other types of rare-cutting endonucleases, such asTAL-nucleases, Zing Finger nucleases or homing endonucleases, withrespect to any types of cells that can be infected by viral vectors.

Cell-Penetrating Peptides Delivery Methods

Further delivery methods have been investigated by the inventors as partof the present invention, in particular the use of cell penetratingpeptides (CPP) for introducing the RNA guide and/or the RNA guidedendonuclease into the T-cells.

Accordingly, the method of the invention may comprise a step ofpreparing composition comprising a cell penetrating peptide linked tothe RNA guide and/or the RNA guided endonuclease. Said CPP, preferablyN-terminal or C-terminal end of CPP can also be associated with themolecules. This association can be covalent or non-covalent. CPPs can besubdivided into two main classes, the first requiring chemical linkagewith the molecule and the second involving the formation of stable,non-covalent complexes. Covalent bonded CPPs form a covalent conjugateby chemical cross-linking (e.g. disulfide bond) or by cloning followedby expression of a CPP-RNA guided endonuclease fusion protein. In apreferred embodiment, said CPP bears a pyrydil disulfide function suchthat the thiol modified molecule forms a disulfide bond with the CPP.Said disulfide bond can be cleaved in particular in a reducingenvironment such as cytoplasm. Non-covalent bonded CPPs arepreferentially amphipathic peptide

Although definition of CPPs is constantly evolving, they are generallydescribed as short peptides of less than 35 amino acids either derivedfrom proteins or from chimeric sequences, which are capable oftransporting polar hydrophilic biomolecules across cell membrane in areceptor independent manner. CPP can be cationic peptides, peptideshaving hydrophobic sequences, amphipatic peptides, peptides havingproline-rich and anti-microbial sequence, and chimeric or bipartitepeptides (Pooga and Langel 2005). In a particular embodiment, cationicCPP can comprise multiple basic of cationic CPPs (e.g., arginine and/orlysine). Preferably, CCP are amphipathic and possess a net positivecharge. CPPs are able to penetrate biological membranes, to trigger themovement of various biomolecules across cell membranes into thecytoplasm and to improve their intracellular routing, therebyfacilitating interactions with the target. Examples of CPP can include:Tat, a nuclear transcriptional activator protein which is a 101 aminoacid protein required for viral replication by human immunodeficiencyvirus type 1 (HIV-1), penetratin, which corresponds to the third helixof the homeoprotein Antennapedia in Drosophilia, Kaposi fibroblastgrowth factor (FGF) signal peptide sequence, integrin 33 signal peptidesequence; Guanine rich-molecular transporters, MPG, pep-1, sweet arrowpeptide, dermaseptins, transportan, pVEC, Human calcitonin, mouse prionprotein (mPrPr), polyarginine peptide Args sequence, VP22 protein fromHerpes Simplex Virus, antimicrobial peptides Buforin I and SynB(US2013/0065314). New variants of CPPs can combine differenttransduction domains.

Non Alloreactive T Cells:

T cell receptors (TCR) are cell surface receptors that participate inthe activation of T cells in response to the presentation of antigen.The TCR is generally made from two chains, alpha and beta, whichassemble to form a heterodimer and associates with the CD3-transducingsubunits to form the T-cell receptor complex present on the cellsurface. Each alpha and beta chain of the TCR consists of animmunoglobulin-like N-terminal variable (V) and constant (C) region, ahydrophobic transmembrane domain, and a short cytoplasmic region. As forimmunoglobulin molecules, the variable region of the alpha and betachains are generated by V(D)J recombination, creating a large diversityof antigen specificities within the population of T cells. However, incontrast to immunoglobulins that recognize intact antigen, T cells areactivated by processed peptide fragments in association with an MHCmolecule, introducing an extra dimension to antigen recognition by Tcells, known as MHC restriction. Recognition of MHC disparities betweenthe donor and recipient through the T cell receptor leads to T cellproliferation and the potential development of GVHD. It has been shownthat normal surface expression of the TCR depends on the coordinatedsynthesis and assembly of all seven components of the complex (Ashwelland Klusner 1990). The inactivation of TCRalpha or TCRbeta can result inthe elimination of the TCR from the surface of T cells preventingrecognition of alloantigen and thus GVHD. However, TCR disruptiongenerally results in the elimination of the CD3 signaling component andalters the means of further T cell expansion.

As one major objective of the present invention is the use of RNA guidedendonuclease as previously described in a method for engineeringnon-alloreactive T-cells, for their use in immunotherapy.

This method more particularly comprises the steps of modifying T-cellsas previously described by inactivating at least one component of theT-cell receptor (TCR) by using a RNA guided endonuclease associated witha specific guide RNA.

Engraftment of allogeneic T-cells is possible by inactivating at leastone gene encoding a TCR component. TCR is rendered not functional in thecells by inactivating TCR alpha gene and/or TCR beta gene(s). TCRinactivation in allogeneic T-cells aims to prevent or reduce GvHD.

By inactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form. In particular embodiments, thegenetic modification of the method relies on the expression, in providedcells to engineer, of the RNA guided endonuclease such that samecatalyzes cleavage in one targeted gene thereby inactivating saidtargeted gene. The nucleic acid strand breaks caused by the endonucleaseare commonly repaired through the distinct mechanisms of homologousrecombination or non-homologous end joining (NHEJ). However, NHEJ is animperfect repair process that often results in changes to the DNAsequence at the site of the cleavage. Mechanisms involve rejoining ofwhat remains of the two DNA ends through direct re-ligation (Critchlowand Jackson 1998) or via the so-called microhomology-mediated endjoining (Betts, Brenchley et al. 2003; Ma, Kim et al. 2003). Repair vianon-homologous end joining (NHEJ) often results in small insertions ordeletions and can be used for the creation of specific gene knockouts.Said modification may be a substitution, deletion, or addition of atleast one nucleotide. Cells in which a cleavage-induced mutagenesisevent, i.e. a mutagenesis event consecutive to an NHEJ event, hasoccurred can be identified and/or selected by well-known method in theart.

The inventors have determined appropriate target sequences within the 3exons encoding TCR, allowing a significant reduction of toxicity, whileretaining cleavage efficiency. The preferred target sequences are notedin Table 2 (+ for lower ratio of TCR negative cells, ++ for intermediateratio, +++ for higher ratio).

TABLE 2 appropriate target sequences for the guideRNA using Cas9 in T-cells Exon SEQ TCR Position StrandTarget genomic sequence ID efficiency Ex1 78 −1 GAGAATCAAAATCGGTGAATAGG6 +++ Ex3 26 1 TTCAAAACCTGTCAGTGATTGGG 7 +++ Ex1 153 1TGTGCTAGACATGAGGTCTATGG 8 +++ Ex3 74 −1 CGTCATGAGCAGATTAAACCCGG 9 +++Ex1 4 −1 TCAGGGTTCTGGATATCTGTGGG 10 +++ Ex1 5 −1 GTCAGGGTTCTGGATATCTGTGG11 +++ Ex3 33 −1 TTCGGAACCCAATCACTGACAGG 12 +++ Ex3 60 −1TAAACCCGGCCACTTTCAGGAGG 13 +++ Ex1 200 −1 AAAGTCAGATTTGTTGCTCCAGG 14 ++Ex1 102 1 AACAAATGTGTCACAAAGTAAGG 15 ++ Ex1 39 −1TGGATTTAGAGTCTCTCAGCTGG 16 ++ Ex1 59 −1 TAGGCAGACAGACTTGTCACTGG 17 ++Ex1 22 −1 AGCTGGTACACGGCAGGGTCAGG 18 ++ Ex1 21 −1GCTGGTACACGGCAGGGTCAGGG 19 ++ Ex1 28 −1 TCTCTCAGCTGGTACACGGCAGG 20 ++Ex3 25 1 TTTCAAAACCTGTCAGTGATTGG 21 ++ Ex3 63 −1 GATTAAACCCGGCCACTTTCAGG22 ++ Ex2 17 −1 CTCGACCAGCTTGACATCACAGG 23 ++ Ex1 32 −1AGAGTCTCTCAGCTGGTACACGG 24 ++ Ex1 27 −1 CTCTCAGCTGGTACACGGCAGGG 25 ++Ex2 12 1 AAGTTCCTGTGATGTCAAGCTGG 26 ++ Ex3 55 1 ATCCTCCTCCTGAAAGTGGCCGG27 ++ Ex3 86 1 TGCTCATGACGCTGCGGCTGTGG 28 ++ Ex1 146 1ACAAAACTGTGCTAGACATGAGG 29 + Ex1 86 −1 ATTTGTTTGAGAATCAAAATCGG 30 + Ex23 −1 CATCACAGGAACTTTCTAAAAGG 31 + Ex2 34 1 GTCGAGAAAAGCTTTGAAACAGG 32 +Ex3 51 −1 CCACTTTCAGGAGGAGGATTCGG 33 + Ex3 18 −1 CTGACAGGTTTTGAAAGTTTAGG34 + Ex2 43 1 AGCTTTGAAACAGGTAAGACAGG 35 + Ex1 236 −1TGGAATAATGCTGTTGTTGAAGG 36 + Ex1 182 1 AGAGCAACAGTGCTGTGGCCTGG 37 + Ex3103 1 CTGTGGTCCAGCTGAGGTGAGGG 38 + Ex3 97 1 CTGCGGCTGTGGTCCAGCTGAGG 39 +Ex3 104 1 TGTGGTCCAGCTGAGGTGAGGGG 40 + Ex1 267 1 CTTCTTCCCCAGCCCAGGTAAGG41 + Ex1 15 −1 ACACGGCAGGGTCAGGGTTCTGG 42 + Ex1 177 1CTTCAAGAGCAACAGTGCTGTGG 43 + Ex1 256 −1 CTGGGGAAGAAGGTGTCTTCTGG 44 + Ex356 1 TCCTCCTCCTGAAAGTGGCCGGG 45 + Ex3 80 1 TTAATCTGCTCATGACGCTGCGG 46 +Ex3 57 −1 ACCCGGCCACTTTCAGGAGGAGG 47 + Ex1 268 1 TTCTTCCCCAGCCCAGGTAAGGG48 + Ex1 266 −1 CTTACCTGGGCTGGGGAAGAAGG 49 + Ex1 262 1GACACCTTCTTCCCCAGCCCAGG 50 + Ex3 102 1 GCTGTGGTCCAGCTGAGGTGAGG 51 + Ex351 1 CCGAATCCTCCTCCTGAAAGTGG 52 +

Method of Engineering Drug-Resistant T-Cells:

To improve cancer therapy and selective engraftment of allogeneicT-cells, drug resistance can be conferred to the engineered T-cells toprotect them from the toxic side effects of chemotherapy orimmunosuppressive agents. Indeed, the inventors have observed that mostpatients were treated with chemotherapy and immune depleting agents as astandard of care, prior to trials involving T-cell immunotherapy. Alsothey found that they could take advantage of these treatments to helpthe selection of the engineered T-cells, either by adding chemotherapydrugs in culture media for expansion of the cells ex-vivo prior totreatment, or by obtaining a selective expansion of the engineeredT-cells in-vivo in patients under chemotherapy or immunosuppressivetreatments.

Also the drug resistance of T-cells also permits their enrichment in orex vivo, as T-cells which express the drug resistance gene, will surviveand multiply relative to drug sensitive cells. In particular, thepresent invention relates to a method of engineering allogeneic and drugresistance T-cells resistant for immunotherapy comprising:

-   -   (a) Providing a T-cell;    -   (b) Selecting at least one drug;    -   (c) Modifying T-cell to confer drug resistance to said T-cell;    -   (d) Expanding said engineered T-cell in the presence of said        drug, and optionally        The preceding steps may be combined with the steps previously        described of:    -   (e) Modifying said T-cell by inactivating at least one gene        encoding a T-cell receptor (TCR) component;    -   (f) Sorting the transformed T-cells, which do not express TCR on        their cell surface;

Thus, according to one aspect of the present invention, the methodcomprises the step of inactivating at least one gene encoding a T-CellReceptor (TCR) component, while further modifying said T-cell to confera resistance to a drug, more particularly a chemotherapy agent. Theresistance to a drug can be conferred to a T-cell by expressing a drugresistance gene. Variant alleles of several genes such as dihydrofolatereductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2),calcineurin or methylguanine transferase (MGMT) have been identified toconfer drug resistance to a cell. Said drug resistance gene can beexpressed in the cell either by introducing a transgene encoding saidgene into the cell or by integrating said drug resistance gene into thegenome of the cell by homologous recombination.

The resistance to a drug can be conferred to a T-cell by inactivatingone or more gene(s) responsible for the cell's sensitivity to the drug(drug sensitizing gene(s)), such as the hypoxanthine-guaninephosphoribosyl transferase (HPRT) gene (Genbank: M26434.1). Inparticular HPRT can be inactivated in engineered T-cells to conferresistance to a cytostatic metabolite, the 6-thioguanine (6TG) which isconverted by HPRT to cytotoxic thioguanine nucleotide and which iscurrently used to treat patients with cancer, in particular leukemias(Hacke, Treger et al. 2013). Another example if the inactivation of theCD3 normally expressed at the surface of the T-cell can conferresistance to anti-CD3 antibodies such as teplizumab.

Otherwise, said drug resistance can be conferred to the T-cell by theexpression of at least one drug resistance gene. Said drug resistancegene refers to a nucleic acid sequence that encodes “resistance” to anagent, such as a chemotherapeutic agent (e.g. methotrexate). In otherwords, the expression of the drug resistance gene in a cell permitsproliferation of the cells in the presence of the agent to a greaterextent than the proliferation of a corresponding cell without the drugresistance gene. A drug resistance gene of the invention can encoderesistance to anti-metabolite, methotrexate, vinblastine, cisplatin,alkylating agents, anthracyclines, cytotoxic antibiotics,anti-immunophilins, their analogs or derivatives, and the like.

Several drug resistance genes have been identified that can potentiallybe used to confer drug resistance to targeted cells (Takebe, Zhao et al.2001; Sugimoto, Tsukahara et al. 2003; Zielske, Reese et al. 2003;Nivens, Felder et al. 2004; Bardenheuer, Lehmberg et al. 2005; Kushman,Kabler et al. 2007).

One example of drug resistance gene can also be a mutant or modifiedform of Dihydrofolate reductase (DHFR). DHFR is an enzyme involved inregulating the amount of tetrahydrofolate in the cell and is essentialto DNA synthesis. Folate analogs such as methotrexate (MTX) inhibit DHFRand are thus used as anti-neoplastic agents in clinic. Different mutantforms of DHFR which have increased resistance to inhibition byanti-folates used in therapy have been described. In a particularembodiment, the drug resistance gene according to the present inventioncan be a nucleic acid sequence encoding a mutant form of human wild typeDHFR (GenBank: AAH71996.1) which comprises at least one mutationconferring resistance to an anti-folate treatment, such as methotrexate.In particular embodiment, mutant form of DHFR comprises at least onemutated amino acid at position G15, L22, F31 or F34, preferably atpositions L22 or F31 ((Schweitzer, Dicker et al. 1990); Internationalapplication WO94/24277; U.S. Pat. No. 6,642,043).

As used herein, “antifolate agent” or “folate analogs” refers to amolecule directed to interfere with the folate metabolic pathway at somelevel. Examples of antifolate agents include, e.g., methotrexate (MTX);aminopterin; trimetrexate (Neutrexin™); edatrexate;N10-propargyl-5,8-dideazafolic acid (CB3717); ZD1694 (Tumodex),5,8-dideazaisofolic acid (IAHQ); 5,10-dideazatetrahydrofolic acid(DDATHF); 5-deazafolic acid; PT523 (N alpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloyl-L-ornithine); 10-ethyl-10-deazaaminopterin (DDATHF,lomatrexol); piritrexim; 10-EDAM; ZD1694; GW1843; Pemetrexate and PDX(10-propargyl-10-deazaaminopterin).

Another example of drug resistance gene can also be a mutant or modifiedform of ionisine-5′-monophosphate dehydrogenase II (IMPDH2), arate-limiting enzyme in the de novo synthesis of guanosine nucleotides.The mutant or modified form of IMPDH2 is a IMPDH inhibitor resistancegene. IMPDH inhibitors can be mycophenolic acid (MPA) or its prodrugmycophenolate mofetil (MMF). The mutant IMPDH2 can comprises at leastone, preferably two mutations in the MAP binding site of the wild typehuman IMPDH2 (NP_000875.2) that lead to a significantly increasedresistance to IMPDH inhibitor. The mutations are preferably at positionsT333 and/or S351 (Yam, Jensen et al. 2006; Sangiolo, Lesnikova et al.2007; Jonnalagadda, Brown et al. 2013). In a particular embodiment, thethreonine residue at position 333 is replaced with an isoleucine residueand the serine residue at position 351 is replaced with a tyrosineresidue.

Another drug resistance gene is the mutant form of calcineurin.Calcineurin (PP2B) is an ubiquitously expressed serine/threonine proteinphosphatase that is involved in many biological processes and which iscentral to T-cell activation. Calcineurin is a heterodimer composed of acatalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB;two isoforms). After engagement of the T-cell receptor, calcineurindephosphorylates the transcription factor NFAT, allowing it totranslocate to the nucleus and active key target gene such as IL2. FK506in complex with FKBP12, or cyclosporine A (CsA) in complex with CyPAblock NFAT access to calcineurin's active site, preventing itsdephosphorylation and thereby inhibiting T-cell activation (Brewin,Mancao et al. 2009). The drug resistance gene of the present inventioncan be a nucleic acid sequence encoding a mutant form of calcineurinresistant to calcineurin inhibitor such as FK506 and/or CsA. In aparticular embodiment, said mutant form can comprise at least onemutated amino acid of the wild type calcineurin heterodimer a atpositions: V314, Y341, M347, T351, W352, L354, K360, preferably doublemutations at positions T351 and L354 or V314 and Y341. Correspondence ofamino acid positions described herein is frequently expressed in termsof the positions of the amino acids of the form of wild-type humancalcineurin heterodimer (GenBank: ACX34092.1).

In another particular embodiment, said mutant form can comprise at leastone mutated amino acid of the wild type calcineurin heterodimer b atpositions: V120, N123, L124 or K125, preferably double mutations atpositions L124 and K125. Correspondence of amino acid positionsdescribed herein is frequently expressed in terms of the positions ofthe amino acids of the form of wild-type human calcineurin heterodimer bpolypeptide (GenBank: ACX34095.1).

Another drug resistance gene is 0(6)-methylguanine methyltransferase(MGMT) encoding human alkyl guanine transferase (hAGT). AGT is a DNArepair protein that confers resistance to the cytotoxic effects ofalkylating agents, such as nitrosoureas and temozolomide (TMZ).6-benzylguanine (6-BG) is an inhibitor of AGT that potentiatesnitrosourea toxicity and is co-administered with TMZ to potentiate thecytotoxic effects of this agent. Several mutant forms of MGMT thatencode variants of AGT are highly resistant to inactivation by 6-BG, butretain their ability to repair DNA damage (Maze, Kurpad et al. 1999). Ina particular embodiment, AGT mutant form can comprise a mutated aminoacid of the wild type AGT position P140 (UniProtKB: P16455).

Another drug resistance gene can be multidrug resistance protein 1(MDR1) gene. This gene encodes a membrane glycoprotein, known asP-glycoprotein (P-GP) involved in the transport of metabolic byproductsacross the cell membrane. The P-Gp protein displays broad specificitytowards several structurally unrelated chemotherapy agents. Thus, drugresistance can be conferred to cells by the expression of nucleic acidsequence that encodes MDR-1 (NP_000918).

Drug resistance gene can also be cytotoxic antibiotics, such as ble geneor mcrA gene. Ectopic expression of ble gene or mcrA in an immune cellgives a selective advantage when exposed to the chemotherapeutic agent,respectively the bleomycine or the mitomycin C.

With respect to the immunosuppressive agents, the present inventionprovides the possible optional steps of:

-   -   (a) Providing a T-cell, preferably from a cell culture or from a        blood sample;    -   (b) Selecting a gene in said T-cell expressing a target for an        immunosuppressive agent;    -   (c) Introducing into said T-cell RNA guided endonuclease able to        selectively inactivate by DNA cleavage, preferably by        double-strand break, said gene encoding a target for said        immunosuppressive agent,    -   (d) Expanding said cells, optionally in presence of said        immunosuppressive agent.

In a more preferred embodiment, said method comprises to inactivate acomponent of the T-cell receptor (TCR).

An immunosuppressive agent is an agent that suppresses immune functionby one of several mechanisms of action. In other words, animmunosuppressive agent is a role played by a compound which isexhibited by a capability to diminish the extent and/or voracity of animmune response. As non-limiting example, an immunosuppressive agent canbe a calcineurin inhibitor, a target of rapamycin, an interleukin-2α-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, aninhibitor of dihydrofolic acid reductase, a corticosteroid or animmunosuppressive antimetabolite. Classical cytotoxic immunosuppressantsact by inhibiting DNA synthesis. Others may act through activation ofT-cells or by inhibiting the activation of helper cells. The methodaccording to the invention allows conferring immunosuppressiveresistance to T cells for immunotherapy by inactivating the target ofthe immunosuppressive agent in T cells. As non limiting examples,targets for immunosuppressive agent can be a receptor for animmunosuppressive agent such as: CD52, glucocorticoid receptor (GR), aFKBP family gene member and a cyclophilin family gene member.

In immunocompetent hosts, allogeneic cells are normally rapidly rejectedby the host immune system. It has been demonstrated that, allogeneicleukocytes present in non-irradiated blood products will persist for nomore than 5 to 6 days. (Boni, Muranski et al. 2008). Thus, to preventrejection of allogeneic cells, the host's immune system must beeffectively suppressed. Glucocorticoidsteroids are widely usedtherapeutically for immunosuppression (Coutinho and Chapman 2011). Thisclass of steroid hormones binds to the glucocorticoid receptor (GR)present in the cytosol of T cells resulting in the translocation intothe nucleus and the binding of specific DNA motifs that regulate theexpression of a number of genes involved in the immunologic process.Treatment of T cells with glucocorticoid steroids results in reducedlevels of cytokine production leading to T cell anergy and interferingin T cell activation. Alemtuzumab, also known as CAMPATH1-H, is ahumanized monoclonal antibody targeting CD52, a 12 amino acidglycosylphosphatidyl-inositol- (GPI) linked glycoprotein (Waldmann andHale 2005). CD52 is expressed at high levels on T and B lymphocytes andlower levels on monocytes while being absent on granulocytes and bonemarrow precursors. Treatment with Alemtuzumab, a humanized monoclonalantibody directed against CD52, has been shown to induce a rapiddepletion of circulating lymphocytes and monocytes. It is frequentlyused in the treatment of T cell lymphomas and in certain cases as partof a conditioning regimen for transplantation. However, in the case ofadoptive immunotherapy the use of immunosuppressive drugs will also havea detrimental effect on the introduced therapeutic T cells. Therefore,to effectively use an adoptive immunotherapy approach in theseconditions, the introduced cells would need to be resistant to theimmunosuppressive treatment.

As a preferred embodiment of the above steps, said gene of step (b),specific for an immunosuppressive treatment, is CD52, and theimmunosuppressive treatment of step (d) comprises a humanized antibodytargeting CD52 antigen. As another embodiment, said gene of step (b),specific for an immunosuppressive treatment, is a glucocorticoidreceptor (GR) and the immunosuppressive treatment of step d) comprises acorticosteroid such as dexamethasone. As another embodiment, said targetgene of step (b), specific for an immunosuppressive treatment, is a FKBPfamily gene member or a variant thereof and the immunosuppressivetreatment of step (d) comprises FK506 also known as Tacrolimus orfujimycin. As another embodiment, said FKBP family gene member is FKBP12or a variant thereof. As another embodiment, said gene of step (b),specific for an immunosuppressive treatment, is a cyclophilin familygene member or a variant thereof and the immunosuppressive treatment ofstep (d) comprises cyclosporine.

In a particular embodiment of the invention, the genetic modificationstep of the method relies on the inactivation of two genes selected fromthe group consisting of CD52 and GR, CD52 and TCR alpha, CDR52 and TCRbeta, GR and TCR alpha, GR and TCR beta, TCR alpha and TCR beta. Inanother embodiment, the genetic modification step of the method relieson the inactivation of more than two genes. The genetic modification ispreferably operated ex-vivo using at least two RNA guides targeting thedifferent genes.

By inactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form.

Engineering Highly Active T Cells for Immunotherapy

In the scope of the present invention is also encompassed an isolated Tcell obtained according to any one of the methods previously described.Said T-cell according to the present invention can be derived from astem cell. The stem cells can be adult stem cells, embryonic stem cells,more particularly non-human stem cells, cord blood stem cells,progenitor cells, bone marrow stem cells, induced pluripotent stemcells, totipotent stem cells or hematopoietic stem cells. Representativehuman cells are CD34+ cells. The T-cells according to the invention canbe selected from the group consisting of inflammatory T-lymphocytes,cytotoxic T-lymphocytes, regulatory T-lymphocytes or helperT-lymphocytes. In another embodiment, said cell can be derived from thegroup consisting of CD4+ T-lymphocytes and CD8+T-lymphocytes. Prior toexpansion and genetic modification of the cells of the invention, asource of cells can be obtained from a subject through a variety ofnon-limiting methods. T cells can be obtained from a number ofnon-limiting sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T cell linesavailable and known to those skilled in the art, may be used. In anotherembodiment, said cell can be derived from a healthy donor, from apatient diagnosed with cancer or from a patient diagnosed with aninfection. In another embodiment, said cell is part of a mixedpopulation of cells which present different phenotypic characteristics.In the scope of the present invention is also encompassed a cell lineobtained from a transformed T-cell according to the method previouslydescribed. Modified cells resistant to an immunosuppressive treatmentand susceptible to be obtained by the previous method are encompassed inthe scope of the present invention.

Immune Check Points

It will be understood by those of ordinary skill in the art, that theterm “immune checkpoints” means a group of molecules expressed by Tcells. These molecules effectively serve as “brakes” to down-modulate orinhibit an immune response. Immune checkpoint molecules include, but arenot limited to Programmed Death 1 (PD-1, also known as PDCD1 or CD279,accession number: NM_005018), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4,also known as CD152, GenBank accession number AF414120.1), LAG3 (alsoknown as CD223, accession number: NM_002286.5), Tim3 (also known asHAVCR2, GenBank accession number: JX049979.1), BTLA (also known asCD272, accession number: NM_181780.3), BY55 (also known as CD160,GenBank accession number: CR541888.1), TIGIT (also known as IVSTM3,accession number: NM_173799), LAIR1 (also known as CD305, GenBankaccession number: CR542051.1, SIGLEC10 (GeneBank accession number:AY358337.1), 2B4 (also known as CD244, accession number:NM_001166664.1), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7,SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD,FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1,IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3,PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3 which directly inhibitimmune cells. For example, CTLA-4 is a cell-surface protein expressed oncertain CD4 and CD8 T cells; when engaged by its ligands (B7-1 and B7-2)on antigen presenting cells, T-cell activation and effector function areinhibited. Thus the present invention relates to a method of engineeringT-cells, especially for immunotherapy, comprising genetically modifyingT-cells by inactivating at least one protein involved in the immunecheckpoint, in particular PD1 and/or CTLA-4 or any immune-checkpointproteins referred to in Table 3.

In a preferred embodiment, at least two genes encoding immune checkpointproteins are inactivated, selected from the group consisting of: CTLA4,PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2, BTLA, CD160, TIGIT,CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A,CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2,SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R,IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3,GUCY1B2, GUCY1B3.

In a further preferred embodiment, the above immune checkpoint proteininactivation is combined with the previous proposed inactivation, inparticular of TCR components.

In another preferred embodiment, said engineered T-cell according to thepresent invention comprises two inactivated genes selected from thegroup consisting of CD52 and GR, CD52 and TCR alpha, CDR52 and TCR beta,GR and TCR alpha, GR and TCR beta, TCR alpha and TCR beta, PD1 and TCRalpha, PD1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG3and TCR alpha, LAG3 and TCR beta, Tim3 and TCR alpha, Tim3 and TCR beta,BTLA and TCR alpha, BTLA and TCR beta, BY55 and TCR alpha, BY55 and TCRbeta, TIGIT and TCR alpha, TIGIT and TCR beta, B7H5 and TCR alpha, B7H5and TCR beta, LAIR1 and TCR alpha, LAIR1 and TCR beta, SIGLEC10 and TCRalpha, SIGLEC10 and TCR beta, 2B4 and TCR alpha, 2B4 and TCR beta and/orexpresses a CAR or a multi-chain CAR.

Engineered T-Cells Expressing Chimeric Antigen Receptors AgainstMalignant Cells

Single-Chain CAR

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific T cells generated ex vivo, is a promising strategy totreat viral infections and cancer. The T cells used for adoptiveimmunotherapy can be generated either by expansion of antigen-specific Tcells or redirection of T cells through genetic engineering (Park,Rosenberg et al. 2011). Transfer of viral antigen specific T cells is awell-established procedure used for the treatment of transplantassociated viral infections and rare viral-related malignancies.Similarly, isolation and transfer of tumor specific T cells has beenshown to be successful in treating melanoma.

TABLE 3 List of genes encoding immune checkpoint proteins. Genes thatcan be inactivated Pathway In the pathway Co-inhibitory CTLA4 (CD152)CTLA4, PPP2CA, PPP2CB, receptors PTPN6, PTPN22 PDCD1 PDCD1 (PD-1, CD279)CD223 (lag3) LAG3 HAVCR2 (tim3) HAVCR2 BTLA(cd272) BTLA CD160(by55)CD160 IgSF family TIGIT CD96 CRTAM LAIR1(cd305) LAIR1 SIGLECs SIGLEC7SIGLEC9 CD244(2b4) CD244 Death receptors TRAIL TNFRSF10B, TNFRSF10A,CASP8, CASP10, CASP3, CASP6, CASP7 FAS FADD, FAS Cytokine TGF-betasignaling TGFBRII, TGFBRI, SMAD2, signalling SMAD3, SMAD4, SMAD10, SKI,SKIL, TGIF1 IL10 signalling IL10RA, IL10RB, HMOX2 IL6 signalling IL6R,IL6ST Prevention of CSK, PAG1 TCR signalling SIT1 Induced Treg inducedTreg FOXP3 Transcription transcription factors PRDM1 (=blimp1, factorscontrolling exhaustion heterozygotes mice control controlling chronicviral infection better exhaustion than wt or conditional KO) BATFHypoxia iNOS induced GUCY1A2, GUCY1A3, mediated guanylated cyclaseGUCY1B2, GUCY1B3 tolerance

Novel specificities in T cells have been successfully generated throughthe genetic transfer of transgenic T cell receptors or chimeric antigenreceptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptorsconsisting of a targeting moiety that is associated with one or moresignaling domains in a single fusion molecule. In general, the bindingmoiety of a CAR consists of an antigen-binding domain of a single-chainantibody (scFv), comprising the light and variable fragments of amonoclonal antibody joined by a flexible linker. Binding moieties basedon receptor or ligand domains have also been used successfully. Thesignaling domains for first generation CARs are derived from thecytoplasmic region of the CD3zeta or the Fc receptor gamma chains. Firstgeneration CARs have been shown to successfully redirect T cellcytotoxicity, however, they failed to provide prolonged expansion andanti-tumor activity in vivo. Signaling domains from co-stimulatorymolecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have beenadded alone (second generation) or in combination (third generation) toenhance survival and increase proliferation of CAR modified T cells.CARs have successfully allowed T cells to be redirected against antigensexpressed at the surface of tumor cells from various malignanciesincluding lymphomas and solid tumors (Jena, Dotti et al. 2010).

The present invention further includes the possibility of expressing achimeric antigen receptor as described in the literature in engineeredT-cells as described herein.

CD19 is an attractive target for immunotherapy because the vast majorityof B-acute lymphoblastic leukemia (B-ALL) uniformly express CD19,whereas expression is absent on non-hematopoietic cells, as well asmyeloid, erythroid, and T cells, and bone marrow stem cells. Clinicaltrials targeting CD19 on B-cell malignancies are underway withencouraging anti-tumor responses. Most infuse T cells geneticallymodified to express a chimeric antigen receptor (CAR) with specificityderived from the scFv region of a CD19-specific mouse monoclonalantibody FMC63 (Nicholson, Lenton et al. 1997; Cooper, Topp et al. 2003;Cooper, Jena et al. 2012) (International application: WO2013/126712).

As an example of single-chain chimeric antigen receptor to be expressedin the genetically engineered T-cells according to the present inventionis a single polypeptide that comprises at least one extracellular ligandbinding domain, a transmembrane domain and at least one signaltransducing domain, wherein said extracellular ligand binding domaincomprises a scFV derived from the specific anti-CD19 monoclonal antibody4G7. Once transduced into the T-cell, for instance by using retroviralor lentiviral transduction, this CAR contributes to the recognition ofCD19 antigen present at the surface of malignant B-cells involved inlymphoma or leukemia.

Other examples of chimeric antigen receptor can also be introduced inthe T-cells according to the present invention, such as CAR bearingantigen receptors directed against multiple myeloma or lymphoblasticleukemia antigen markers, such as TNFRSF17 (UNIPROT Q02223), SLAMF7(UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4),KAMP3, ITGA8 (UNIPROT P53708), and FCRL5 (UNIPROT Q68SN8).

As further examples, the antigen of the target can be from any clusterof differentiation molecules (e.g. CD16, CD64, CD78, CD96, CLL1, CD116,CD117, CD71, CD45, CD71, CD123 and CD138), a tumor-associated surfaceantigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA),epithelial cell adhesion molecule (EpCAM), epidermal growth factorreceptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40,disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72,glycosphingolipids, glioma-associated antigen, 3-human chorionicgonadotropin, alphafetoprotein (AFP), lectin-reactive AFP,thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF,prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53,prostein, PSMA, surviving and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22,insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, amajor histocompatibility complex (MHC) molecule presenting atumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromalantigens, the extra domain A (EDA) and extra domain B (EDB) offibronectin and the A1 domain of tenascin-C(TnC A1) and fibroblastassociated protein (fap); a lineage-specific or tissue specific antigensuch as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4,B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a majorhistocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), or avirus-specific surface antigen such as an HIV-specific antigen (such asHIV gp120); an EBV-specific antigen, a CMV-specific antigen, aHPV-specific antigen, a Lasse Virus-specific antigen, an InfluenzaVirus-specific antigen as well as any derivate or variant of thesesurface markers. Antigens are not necessarily surface marker antigensbut can be also endogenous small antigens presented by HLA class I atthe surface of the cells.

Multi-Subunit CAR

Chimeric antigen receptors from the prior art introduced in T-cells havebeen formed of single chain polypeptides that necessitate serialappending of signaling domains. However, by moving signaling domainsfrom their natural juxtamembrane position may interfere with theirfunction. To overcome this drawback, the applicant recently designed amulti-chain CAR derived from FcεRI to allow normal juxtamembraneposition of all relevant signaling domains. In this new architecture,the high affinity IgE binding domain of FcεRI alpha chain is replaced byan extracellular ligand-binding domain such as scFv to redirect T-cellspecificity against cell targets and the N and/or C-termini tails ofFcεRI beta chain are used to place costimulatory signals in normaljuxtamembrane positions.

Accordingly, the CAR expressed by the engineered T-cell according to theinvention can be a multi-chain chimeric antigen receptor (CAR)particularly adapted to the production and expansion of engineeredT-cells of the present invention. Such multi-chain CARs comprise atleast two of the following components:

-   -   a) one polypeptide comprising the transmembrembrane domain of        FcεRI alpha chain and an extracellular ligand-binding domain,    -   b) one polypeptide comprising a part of N- and C-terminal        cytoplasmic tail and the transmembrane domain of FcεRI beta        chain and/or    -   c) at least two polypeptides comprising each a part of        intracytoplasmic tail and the transmembrane domain of FcεRI        gamma chain, whereby different polypeptides multimerize together        spontaneously to form dimeric, trimeric or tetrameric CAR.

According to such architectures, ligands binding domains and signalingdomains are born on separate polypeptides. The different polypeptidesare anchored into the membrane in a close proximity allowinginteractions with each other. In such architectures, the signaling andco-stimulatory domains can be in juxtamembrane positions (i.e. adjacentto the cell membrane on the internal side of it), which is deemed toallow improved function of co-stimulatory domains. The multi-subunitarchitecture also offers more flexibility and possibilities of designingCARs with more control on T-cell activation. For instance, it ispossible to include several extracellular antigen recognition domainshaving different specificity to obtain a multi-specific CARarchitecture. It is also possible to control the relative ratio betweenthe different subunits into the multi-chain CAR. This type ofarchitecture has been recently described by the applicant inPCT/US2013/058005.

The assembly of the different chains as part of a single multi-chain CARis made possible, for instance, by using the different alpha, beta andgamma chains of the high affinity receptor for IgE (FcεRI) (Metzger,Alcaraz et al. 1986) to which are fused the signaling and co-stimulatorydomains. The gamma chain comprises a transmembrane region andcytoplasmic tail containing one immunoreceptor tyrosine-based activationmotif (ITAM) (Cambier 1995).

The multi-chain CAR can comprise several extracellular ligand-bindingdomains, to simultaneously bind different elements in target therebyaugmenting immune cell activation and function. In one embodiment, theextracellular ligand-binding domains can be placed in tandem on the sametransmembrane polypeptide, and optionally can be separated by a linker.In another embodiment, said different extracellular ligand-bindingdomains can be placed on different transmembrane polypeptides composingthe multi-chain CAR. In another embodiment, the present inventionrelates to a population of multi-chain CARs comprising each onedifferent extracellular ligand binding domains. In a particular, thepresent invention relates to a method of engineering immune cellscomprising providing an immune cell and expressing at the surface ofsaid cell a population of multi-chain CAR each one comprising differentextracellular ligand binding domains. In another particular embodiment,the present invention relates to a method of engineering an immune cellcomprising providing an immune cell and introducing into said cellpolynucleotides encoding polypeptides composing a population ofmulti-chain CAR each one comprising different extracellular ligandbinding domains. In a particular embodiment the method of engineering animmune cell comprises expressing at the surface of the cell at least apart of FcεRI beta and/or gamma chain fused to a signal-transducingdomain and several part of FcεRI alpha chains fused to differentextracellular ligand binding domains. In a more particular embodiment,said method comprises introducing into said cell at least onepolynucleotide which encodes a part of FcεRI beta and/or gamma chainfused to a signal-transducing domain and several FcεRI alpha chainsfused to different extracellular ligand biniding domains. By populationof multi-chain CARs, it is meant at least two, three, four, five, six ormore multi-chain CARs each one comprising different extracellular ligandbinding domains. The different extracellular ligand binding domainsaccording to the present invention can preferably simultaneously binddifferent elements in target thereby augmenting immune cell activationand function.

The present invention also relates to an isolated immune cell whichcomprises a population of multi-chain CARs each one comprising differentextracellular ligand binding domains.

The signal transducing domain or intracellular signaling domain of themulti-chain CAR of the invention is responsible for intracellularsignaling following the binding of extracellular ligand binding domainto the target resulting in the activation of the immune cell and immuneresponse. In other words, the signal transducing domain is responsiblefor the activation of at least one of the normal effector functions ofthe immune cell in which the multi-chain CAR is expressed. For example,the effector function of a T cell can be a cytolytic activity or helperactivity including the secretion of cytokines.

In the present application, the term “signal transducing domain” refersto the portion of a protein which transduces the effector signalfunction signal and directs the cell to perform a specialized function.

Preferred examples of signal transducing domain for use in single ormulti-chain CAR can be the cytoplasmic sequences of the Fc receptor or Tcell receptor and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivate or variant of these sequences and any synthetic sequence thatas the same functional capability. Signal transduction domain comprisestwo distinct classes of cytoplasmic signaling sequence, those thatinitiate antigen-dependent primary activation, and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal. Primary cytoplasmic signaling sequence can comprise signalingmotifs which are known as immunoreceptor tyrosine-based activationmotifs of ITAMs. ITAMs are well defined signaling motifs found in theintracytoplasmic tail of a variety of receptors that serve as bindingsites for syk/zap70 class tyrosine kinases. Examples of ITAM used in theinvention can include as non-limiting examples those derived fromTCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon,CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, thesignaling transducing domain of the multi-chain CAR can comprise theCD3zeta signaling domain, or the intracytoplasmic domain of the FcεRIbeta or gamma chains.

In particular embodiment the signal transduction domain of themulti-chain CAR of the present invention comprises a co-stimulatorysignal molecule. A co-stimulatory molecule is a cell surface moleculeother than an antigen receptor or their ligands that is required for anefficient immune response.

Ligand binding-domains can be any antigen receptor previously used, andreferred to, with respect to single-chain CAR referred to in theliterature, in particular scFv from monoclonal antibodies.

Bispecific or multi-specific CARs as described in WO 2014/4011988 alsoenter the scope of the present invention.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells, the Tcells can be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005. T cells can be expanded in vitro or in vivo.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3 TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells.

In particular, T cell populations may be stimulated in vitro such as bycontact with an anti-CD3 antibody, or antigen-binding fragment thereof,or an anti-CD2 antibody immobilized on a surface, or by contact with aprotein kinase C activator (e.g., bryostatin) in conjunction with acalcium ionophore. For co-stimulation of an accessory molecule on thesurface of the T cells, a ligand that binds the accessory molecule isused. For example, a population of T cells can be contacted with ananti-CD3 antibody and an anti-CD28 antibody, under conditionsappropriate for stimulating proliferation of the T cells. To stimulateproliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3antibody and an anti-CD28 antibody. For example, the agents providingeach signal may be in solution or coupled to a surface. As those ofordinary skill in the art can readily appreciate, the ratio of particlesto cells may depend on particle size relative to the target cell. Infurther embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. Cell surface proteinsmay be ligated by allowing paramagnetic beads to which anti-CD3 andanti-CD28 are attached (3×28 beads) to contact the T cells. In oneembodiment the cells (for example, 4 to 10 T cells) and beads (forexample, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of1:1) are combined in a buffer, preferably PBS (without divalent cationssuch as, calcium and magnesium). Again, those of ordinary skill in theart can readily appreciate any cell concentration may be used. Themixture may be cultured for several hours (about 3 hours) to about 14days or any hourly integer value in between. In another embodiment, themixture may be cultured for 21 days. Conditions appropriate for T cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4,1L-7, GM-CSF, -10, -2, 1L-15, TGFp, and TNF- or any other additives forthe growth of cells known to the skilled artisan. Other additives forthe growth of cells include, but are not limited to, surfactant,plasmanate, and reducing agents such as N-acetyl-cysteine and2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM,F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodiumpyruvate, and vitamins, either serum-free or supplemented with anappropriate amount of serum (or plasma) or a defined set of hormones,and/or an amount of cytokine(s) sufficient for the growth and expansionof T cells. Antibiotics, e.g., penicillin and streptomycin, are includedonly in experimental cultures, not in cultures of cells that are to beinfused into a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% C02). T cells that havebeen exposed to varied stimulation times may exhibit differentcharacteristics

In another particular embodiment, said cells can be expanded byco-culturing with tissue or cells. Said cells can also be expanded invivo, for example in the subject's blood after administrating said cellinto the subject.

Therapeutic Applications

The T-cells obtainable by the different methods described above areintended to be used as a medicament for treating, among others, cancer,infections or immune diseases in a patient in need thereof.

Said treatment can be ameliorating, curative or prophylactic. It may beeither part of an autologous immunotherapy or part of an allogenicimmunotherapy treatment. By autologous, it is meant that cells, cellline or population of cells used for treating patients are originatingfrom said patient or from a Human Leucocyte Antigen (HLA) compatibledonor. By allogeneic is meant that the cells or population of cells usedfor treating patients are not originating from said patient but from adonor.

The invention is particularly suited for allogenic immunotherapy,insofar as it enables the transformation of T-cells, typically obtainedfrom donors, into non-alloreactive cells. This may be done understandard protocols and reproduced as many times as needed. The resultedmodified T cells may be pooled and administrated to one or severalpatients, being made available as an “off the shelf” therapeuticproduct. Cells that can be used with the disclosed methods are describedin the previous section.

Said treatments are primarily to treat patients diagnosed with cancer,viral infection, autoimmune disorders or Graft versus Host Disease(GvHD). Cancers are preferably leukemias and lymphomas, which haveliquid tumors, but may also concern solid tumors. Types of cancers to betreated with the CARs of the invention include, but are not limited to,carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoidmalignancies, benign and malignant tumors, and malignancies e.g.,sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatrictumors/cancers are also included.

The present T-cells, when armed with specific CARs directed againstpatient's own immune cells, allow the inhibition or regulation of saidcells, which is a key step for treating auto-immune disease, such asrheumatoid polyarthritis, systemic lupus erythematosus, Sjogren'ssyndrome, scleroderma, fibromyalgia, myositis, ankylosing spondylitis,insulin dependent diabetes of type I, Hashimoto's thyroiditis, Addison'sdisease, Crohn's disease, Celiac's disease, amyotrophic lateralsclerosis (ALS) and multiple sclerosis (MS).

The treatment can take place in combination with one or more therapiesselected from the group of antibodies therapy, chemotherapy, cytokinestherapy, dendritic cell therapy, gene therapy, hormone therapy, laserlight therapy and radiation therapy.

According to a preferred embodiment of the invention, said treatment areparticularly suited for patients undergoing immunosuppressive orchemotherapy treatments. Indeed, the present invention preferably relieson cells or population of cells, which have been made resistant to atleast one immunosuppressive agent due to the inactivation of a geneencoding a receptor for such immunosuppressive agent. In this aspect,the immunosuppressive treatment should help the selection and expansionof the T-cells according to the invention within the patient.

According to one embodiment, said T cells of the invention can undergorobust in vivo T cell expansion upon administration to a patient, andcan persist in the body fluids for an extended amount of time,preferably for a week, more preferably for 2 weeks, even more preferablyfor at least one month. Although the T-cells according to the inventionare expected to persist during these periods, their life span into thepatient's body are intended not to exceed a year, preferably 6 months,more preferably 2 months, and even more preferably one month.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includingby aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The compositions described herein may beadministered to a patient subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous orintralymphatic injection, or intraperitoneally. In one embodiment, thecell compositions of the present invention are preferably administeredby intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 10⁴-10⁹ cells per kg body weight, preferably 105to 10⁶ cells/kg body weight including all integer values of cell numberswithin those ranges. The cells or population of cells can beadministrated in one or more doses. In another embodiment, saideffective amount of cells are administrated as a single dose. In anotherembodiment, said effective amount of cells are administrated as morethan one dose over a period time. Timing of administration is within thejudgment of managing physician and depends on the clinical condition ofthe patient. The cells or population of cells may be obtained from anysource, such as a blood bank or a donor. While individual needs vary,determination of optimal ranges of effective amounts of a given celltype for a particular disease or conditions within the skill of the art.An effective amount means an amount which provides a therapeutic orprophylactic benefit. The dosage administrated will be dependent uponthe age, health and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment and the nature of the effectdesired.

In another embodiment, said effective amount of cells or compositioncomprising those cells are administrated parenterally. Saidadministration can be an intravenous administration. Said administrationcan be directly done by injection within a tumor.

In certain embodiments of the present invention, cells are administeredto a patient in conjunction with (e.g., before, simultaneously orfollowing) any number of relevant treatment modalities, including butnot limited to treatment with agents such as antiviral therapy,cidofovir and interleukin-2, Cytarabine (also known as ARA-C) ornataliziimab treatment for MS patients or efaliztimab treatment forpsoriasis patients or other treatments for PML patients. In furtherembodiments, the T cells of the invention may be used in combinationwith chemotherapy, radiation, immunosuppressive agents, such ascyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAMPATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1 1; Henderson et al., Immun.73:316-321, 1991; Bierer et al., Citrr. Opin. mm n. 5:763-773, 93). In afurther embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH, In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery. Said modified cells obtained by any one of themethods described here can be used in a particular aspect of theinvention for treating patients in need thereof against Host versusGraft (HvG) rejection and Graft versus Host Disease (GvHD); therefore inthe scope of the present invention is a method of treating patients inneed thereof against Host versus Graft (HvG) rejection and Graft versusHost Disease (GvHD) comprising treating said patient by administering tosaid patient an effective amount of modified cells comprisinginactivated TCR alpha and/or TCR beta genes.

Other Definitions

-   -   Amino acid residues in a polypeptide sequence are designated        herein according to the one-letter code, in which, for example,        Q means Gin or Glutamine residue, R means Arg or Arginine        residue and D means Asp or Aspartic acid residue.    -   Amino acid substitution means the replacement of one amino acid        residue with another, for instance the replacement of an        Arginine residue with a Glutamine residue in a peptide sequence        is an amino acid substitution.    -   Nucleotides are designated as follows: one-letter code is used        for designating the base of a nucleoside: a is adenine, t is        thymine, c is cytosine, and g is guanine. For the degenerated        nucleotides, r represents g or a (purine nucleotides), k        represents g or t, s represents g or c, w represents a or t, m        represents a or c, y represents t or c (pyrimidine nucleotides),        d represents g, a or t, v represents g, a or c, b represents g,        t or c, h represents a, t or c, and n represents g, a, t or c.    -   “As used herein, “nucleic acid” or “polynucleotides” refers to        nucleotides and/or polynucleotides, such as deoxyribonucleic        acid (DNA) or ribonucleic acid (RNA), oligonucleotides,        fragments generated by the polymerase chain reaction (PCR), and        fragments generated by any of ligation, scission, endonuclease        action, and exonuclease action. Nucleic acid molecules can be        composed of monomers that are naturally-occurring nucleotides        (such as DNA and RNA), or analogs of naturally-occurring        nucleotides (e.g., enantiomeric forms of naturally-occurring        nucleotides), or a combination of both. Modified nucleotides can        have alterations in sugar moieties and/or in pyrimidine or        purine base moieties. Sugar modifications include, for example,        replacement of one or more hydroxyl groups with halogens, alkyl        groups, amines, and azido groups, or sugars can be        functionalized as ethers or esters. Moreover, the entire sugar        moiety can be replaced with sterically and electronically        similar structures, such as aza-sugars and carbocyclic sugar        analogs. Examples of modifications in a base moiety include        alkylated purines and pyrimidines, acylated purines or        pyrimidines, or other well-known heterocyclic substitutes.        Nucleic acid monomers can be linked by phosphodiester bonds or        analogs of such linkages. Nucleic acids can be either single        stranded or double stranded.    -   by “polynucleotide successively comprising a first region of        homology to sequences upstream of said double-stranded break, a        sequence to be inserted in the genome of said cell and a second        region of homology to sequences downstream of said        double-stranded break” it is intended to mean a DNA construct or        a matrix comprising a first and second portion that are        homologous to regions 5′ and 3′ of a DNA target in situ. The DNA        construct also comprises a third portion positioned between the        first and second portion which comprise some homology with the        corresponding DNA sequence in situ or alternatively comprise no        homology with the regions 5′ and 3′ of the DNA target in situ.        Following cleavage of the DNA target, a homologous recombination        event is stimulated between the genome containing the targeted        gene comprised in the locus of interest and this matrix, wherein        the genomic sequence containing the DNA target is replaced by        the third portion of the matrix and a variable part of the first        and second portions of said matrix.    -   by “DNA target”, “DNA target sequence”, “target DNA sequence”,        “nucleic acid target sequence”, “target sequence”, or        “processing site” is intended a polynucleotide sequence that can        be targeted and processed by a rare-cutting endonuclease        according to the present invention. These terms refer to a        specific DNA location, preferably a genomic location in a cell,        but also a portion of genetic material that can exist        independently to the main body of genetic material such as        plasmids, episomes, virus, transposons or in organelles such as        mitochondria as non-limiting example. As non-limiting examples        of RNA guided target sequences, are those genome sequences that        can hybridize the guide RNA which directs the RNA guided        endonuclease to a desired locus.    -   By “delivery vector” or “delivery vectors” is intended any        delivery vector which can be used in the present invention to        put into cell contact (i.e “contacting”) or deliver inside cells        or subcellular compartments (i.e “introducing”) agents/chemicals        and molecules (proteins or nucleic acids) needed in the present        invention. It includes, but is not limited to liposomal delivery        vectors, viral delivery vectors, drug delivery vectors, chemical        carriers, polymeric carriers, lipoplexes, polyplexes,        dendrimers, microbubbles (ultrasound contrast agents),        nanoparticles, emulsions or other appropriate transfer vectors.        These delivery vectors allow delivery of molecules, chemicals,        macromolecules (genes, proteins), or other vectors such as        plasmids, or penetrating peptides. In these later cases,        delivery vectors are molecule carriers.    -   The terms “vector” or “vectors” refer to a nucleic acid molecule        capable of transporting another nucleic acid to which it has        been linked. A “vector” in the present invention includes, but        is not limited to, a viral vector, a plasmid, a RNA vector or a        linear or circular DNA or RNA molecule which may consists of a        chromosomal, non-chromosomal, semi-synthetic or synthetic        nucleic acids. Preferred vectors are those capable of autonomous        replication (episomal vector) and/or expression of nucleic acids        to which they are linked (expression vectors). Large numbers of        suitable vectors are known to those of skill in the art and        commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e. g.adenoassociated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabiesand vesicular stomatitis virus), paramyxovirus (e. g. measles andSendai), positive strand RNA viruses such as picornavirus andalphavirus, and double-stranded DNA viruses including adenovirus,herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include: avian leukosis-sarcoma, mammalianC-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, In Fundamental Virology, Third Edition, B. N. Fields, etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

-   -   By “lentiviral vector” is meant HIV-Based lentiviral vectors        that are very promising for gene delivery because of their        relatively large packaging capacity, reduced immunogenicity and        their ability to stably transduce with high efficiency a large        range of different cell types. Lentiviral vectors are usually        generated following transient transfection of three (packaging,        envelope and transfer) or more plasmids into producer cells.        Like HIV, lentiviral vectors enter the target cell through the        interaction of viral surface glycoproteins with receptors on the        cell surface. On entry, the viral RNA undergoes reverse        transcription, which is mediated by the viral reverse        transcriptase complex. The product of reverse transcription is a        double-stranded linear viral DNA, which is the substrate for        viral integration in the DNA of infected cells. By “integrative        lentiviral vectors (or LV)”, is meant such vectors as non        limiting example, that are able to integrate the genome of a        target cell. At the opposite by “non integrative lentiviral        vectors (or NILV)” is meant efficient gene delivery vectors that        do not integrate the genome of a target cell through the action        of the virus integrase.    -   Delivery vectors and vectors can be associated or combined with        any cellular permeabilization techniques such as sonoporation or        electroporation or derivatives of these techniques.    -   By cell or cells is intended any eukaryotic living cells,        primary cells and cell lines derived from these organisms for in        vitro cultures.    -   By “primary cell” or “primary cells” are intended cells taken        directly from living tissue (i.e. biopsy material) and        established for growth in vitro, that have undergone very few        population doublings and are therefore more representative of        the main functional components and characteristics of tissues        from which they are derived from, in comparison to continuous        tumorigenic or artificially immortalized cell lines.

As non-limiting examples cell lines can be selected from the groupconsisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells,U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLacells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4cells.

All these cell lines can be modified by the method of the presentinvention to provide cell line models to produce, express, quantify,detect, study a gene or a protein of interest; these models can also beused to screen biologically active molecules of interest in research andproduction and various fields such as chemical, biofuels, therapeuticsand agronomy as non-limiting examples.

-   -   by “mutation” is intended the substitution, deletion, insertion        of up to one, two, three, four, five, six, seven, eight, nine,        ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty        five, thirty, forty, fifty, or more nucleotides/amino acids in a        polynucleotide (cDNA, gene) or a polypeptide sequence. The        mutation can affect the coding sequence of a gene or its        regulatory sequence. It may also affect the structure of the        genomic sequence or the structure/stability of the encoded mRNA.    -   by “variant(s)”, it is intended a repeat variant, a variant, a        DNA binding variant, a TALE-nuclease variant, a polypeptide        variant obtained by mutation or replacement of at least one        residue in the amino acid sequence of the parent molecule.    -   by “functional variant” is intended a catalytically active        mutant of a protein or a protein domain; such mutant may have        the same activity compared to its parent protein or protein        domain or additional properties, or higher or lower activity.    -   By “gene” is meant the basic unit of heredity, consisting of a        segment of DNA arranged in a linear manner along a chromosome,        which codes for a specific protein or segment of protein. A gene        typically includes a promoter, a 5′ untranslated region, one or        more coding sequences (exons), optionally introns, a 3′        untranslated region. The gene may further comprise a terminator,        enhancers and/or silencers.    -   As used herein, the term “locus” is the specific physical        location of a DNA sequence (e.g. of a gene) on a chromosome. The        term “locus” can refer to the specific physical location of a        rare-cutting endonuclease target sequence on a chromosome. Such        a locus can comprise a target sequence that is recognized and/or        cleaved by a rare-cutting endonuclease according to the        invention. It is understood that the locus of interest of the        present invention can not only qualify a nucleic acid sequence        that exists in the main body of genetic material (i.e. in a        chromosome) of a cell but also a portion of genetic material        that can exist independently to said main body of genetic        material such as plasmids, episomes, virus, transposons or in        organelles such as mitochondria as non-limiting examples.    -   The term “cleavage” refers to the breakage of the covalent        backbone of a polynucleotide. Cleavage can be initiated by a        variety of methods including, but not limited to, enzymatic or        chemical hydrolysis of a phosphodiester bond. Both        single-stranded cleavage and double-stranded cleavage are        possible, and double-stranded cleavage can occur as a result of        two distinct single-stranded cleavage events. Double stranded        DNA, RNA, or DNA/RNA hybrid cleavage can result in the        production of either blunt ends or staggered ends.    -   By “fusion protein” is intended the result of a well-known        process in the art consisting in the joining of two or more        genes which originally encode for separate proteins or part of        them, the translation of said “fusion gene” resulting in a        single polypeptide with functional properties derived from each        of the original proteins.    -   “identity” refers to sequence identity between two nucleic acid        molecules or polypeptides. Identity can be determined by        comparing a position in each sequence which may be aligned for        purposes of comparison. When a position in the compared sequence        is occupied by the same base, then the molecules are identical        at that position. A degree of similarity or identity between        nucleic acid or amino acid sequences is a function of the number        of identical or matching nucleotides at positions shared by the        nucleic acid sequences. Various alignment algorithms and/or        programs may be used to calculate the identity between two        sequences, including FASTA, or BLAST which are available as a        part of the GCG sequence analysis package (University of        Wisconsin, Madison, Wis.), and can be used with, e.g., default        setting. For example, polypeptides having at least 70%, 85%,        90%, 95%, 98% or 99% identity to specific polypeptides described        herein and preferably exhibiting substantially the same        functions, as well as polynucleotide encoding such polypeptides,        are contemplated.    -   “signal-transducing domain” or “co-stimulatory ligand” refers to        a molecule on an antigen presenting cell that specifically binds        a cognate co-stimulatory molecule on a T-cell, thereby providing        a signal which, in addition to the primary signal provided by,        for instance, binding of a TCR/CD3 complex with an MHC molecule        loaded with peptide, mediates a T cell response, including, but        not limited to, proliferation activation, differentiation and        the like. A co-stimulatory ligand can include but is not limited        to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L,        inducible costimulatory ligand (ICOS-L), intercellular adhesion        molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB,        HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist        or antibody that binds Toll ligand receptor and a ligand that        specifically binds with B7-H3. A co-stimulatory ligand also        encompasses, inter alia, an antibody that specifically binds        with a co-stimulatory molecule present on a T cell, such as but        not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS,        lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,        LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.    -   A “co-stimulatory molecule” refers to the cognate binding        partner on a Tcell that specifically binds with a co-stimulatory        ligand, thereby mediating a co-stimulatory response by the cell,        such as, but not limited to proliferation. Co-stimulatory        molecules include, but are not limited to an MHC class I        molecule, BTLA and Toll ligand receptor.    -   A “co-stimulatory signal” as used herein refers to a signal,        which in combination with primary signal, such as TCR/CD3        ligation, leads to T cell proliferation and/or upregulation or        downregulation of key molecules.    -   “bispecific antibody” refers to an antibody that has binding        sites for two different antigens within a single antibody        molecule. It will be appreciated by those skilled in the art        that other molecules in addition to the canonical antibody        structure may be constructed with two binding specificities. It        will further be appreciated that antigen binding by bispecific        antibodies may be simultaneous or sequential. Bispecific        antibodies can be produced by chemical techniques (see e.g.,        Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78, 5807), by        “polydoma” techniques (See U.S. Pat. No. 4,474,893) or by        recombinant DNA techniques, which all are known per se. As a        non-limiting example, each binding domain comprises at least one        variable region from an antibody heavy chain (“VH or H region”),        wherein the VH region of the first binding domain specifically        binds to the lymphocyte marker such as CD3, and the VH region of        the second binding domain specifically binds to tumor antigen.    -   The term “extracellular ligand-binding domain” as used herein is        defined as an oligo- or polypeptide that is capable of binding a        ligand. Preferably, the domain will be capable of interacting        with a cell surface molecule. For example, the extracellular        ligand-binding domain may be chosen to recognize a ligand that        acts as a cell surface marker on target cells associated with a        particular disease state. Thus examples of cell surface markers        that may act as ligands include those associated with viral,        bacterial and parasitic infections, autoimmune disease and        cancer cells.    -   The term “subject” or “patient” as used herein includes all        members of the animal kingdom including non-human primates and        humans.    -   The above written description of the invention provides a manner        and process of making and using it such that any person skilled        in this art is enabled to make and use the same, this enablement        being provided in particular for the subject matter of the        appended claims, which make up a part of the original        description.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

EXAMPLES Example 1: General Method to Engineer Human Allogeneic Cellsfor Immunotherapy

For a better understanding of the invention, method to engineer humanallogenic cells for immunotherapy is illustrated in FIG. 4. The methodcomprising a combination of one or several of the following steps:

-   -   1. Providing T-cells from a cell culture or from a blood sample        from one individual patient or from blood bank and activating        said T cells using anti-CD3/C28 activator beads. The beads        provide both the primary and co-stimulatory signals that are        required for activation and expansion of T cells.    -   2. Transducing said cells with multi-chain CARs allow        redirecting T cells against antigens expressed at the surface of        target cells from various malignancies including lymphomas and        solid tumors, in particular against the antigen CD19. To improve        the function of co-stimulatory domain, the inventors have        designed a multi-chain CAR derived from FcεRI as previously        described. Transduction can be realized before or after the gene        inactivations using the RNA guided endonuclease.    -   3. Engineering non alloreactive and optionally resistant T        cells:        -   a) It is possible to Inactivate TCR alpha in said cells to            eliminate the TCR from the surface of the cell and prevent            recognition of host tissue as foreign by TCR of allogenic            and thus to avoid GvHD.        -   b) It is also possible to inactive one gene encoding target            for an immunosuppressive agent or a chemotherapy drug to            render said cells resistant to immunosuppressive or            chemotherapy treatment to prevent graft rejection without            affecting transplanted T cells. In this example, target of            immunosuppressive agents is CD52 and immunosuppressive agent            is a humanized monoclonal anti-CD52 antibody.    -    As shown below by the inventors, the use of RNA guided        endonuclease is particularly advantageous to achieve double gene        inactivations in T-cells by merely introducing two distinct        guide-RNA (gRNA), each targeting one gene, such as TCRalpha and        CD52 genes. This can be done by electoporating T cells with mRNA        encoding Cas9 and simultaneously transcription of DNA plasmids        coding for the specific gRNA. Alternatively, the gRNA may be        transcribed from DNA sequences stably integrated into the genome        using retroviral vectors. It has been found by the inventors        that transiently expressing Cas9 from mRNA resulted into high        transformation rate and was less harmful to T-cells. Then,        inactivated T cells are sorted using magnetic beads. For        example, T cells still expressing the targeted gene (e.g. CD52)        can be removed by fixation on a solid surface, and inactivated        cells are not exposed of the stress of being passed through a        column. This gentle method increases the concentration of        properly engineered T-cells.    -   4. Expansion in vitro of engineered T-cells prior to        administration to a patient or in vivo following administration        to a patient through stimulation of CD3 complex. Before        administration step, patients can be subjected to an        immunosuppressive treatment such as CAMPATH1-H, a humanized        monoclonal anti-CD52 antibody.    -   5. Optionally exposed said cells with bispecific antibodies ex        vivo prior to administration to a patient or in vivo following        administration to a patient to bring the engineered cells into        proximity to a target antigen.

Example 2: Specific Inactivation of TCR and CD52 Genes in the T-Cells

1—CAS9 and gRNA Plasmids Construction

The sequence encoding the Cas9 from S. pyogenes was synthesized de novo(GeneCust) and flanked by 3×NLS and a HA tag at the C-terminus(pCLS22972 (SEQ ID NO. 53)). Sequences encoding the gRNA targeting therespective 20 bp sequences (5′ to 3′) in the TCRa gene (SEQ ID NO. 54)and the CD52 gene (SEQ ID NO. 55) were cloned in pUC57 derived plasmiddownstream an U6 promoter leading to respectively pCLS24029 (SEQ ID NO.56) and pCLS24027 (SEQ ID NO. 57) plasmid vectors.

2—CAS9 mRNA Synthesis

mRNA encoding CAS9 was produced and polyadenylated using the mMessagemMachine T7 Ultra kit (Life technologies) following the manufacturer'sinstructions. The RNA was subsequently purified with RNeasy columns(Qiagen), eluted in electroporation buffer, and quantified (NanodropND-1000 spectrophotometer) by measuring absorbance at 260 nm. Quality ofthe RNA was verified on a denaturing formaldehyde/MOPS agarose gel.

3—Transfections

T lymphocytes cells were transfected by electrotransfer of mRNA codingfor the CAS9 and plasmids coding for gRNA using an AgilePulse MAX system(Harvard Apparatus) 4 days after activation (Dynabeads® HumanT-Activator CD3/CD28, Life technologies). Briefly, T-cells werepreactivated several days (3-5) pre-transfection with anti-CD3/CD28coated beads and IL2. One day before electroporation, culture medium waschanged and cells were seeded at 10⁶ cells/ml; 24 h later, T-cells werewashed in cytoporation medium T (Harvard Apparatus), activation beadswere removed by decanting the T-cells suspension from a tube inserted inan EasySep magnet (StemCell technologies). T-cells were then pelleted,resuspended in cytoporation medium T at 25×10⁶ cells/ml. 5×10⁶ cellswere mixed with 10 μg of the mRNA encoding the CAS9 and the plasmidsencoding the gRNA targeting TCRa or into a 0.4 cm cuvette. Theelectroporation consisted of two 0.1 ms pulses at 1200 V followed byfour 0.2 ms pulses at 130 V. Following electroporation, T-cells werediluted into culture medium and incubated at 37° C./5% CO₂ for 24 hoursbefore another medium change. 3 days post electroporation, T-cells werestained with a fixable viability dye eFluor-780 and a PE orAPC-conjugated goat anti mouse IgG F(ab′)2 fragment specific to assessthe cell surface expression of the TCRa or the CD52 respectively.Knock-out of either the TCRa or CD52 genes was analyzed (MACSQuant) byflow cytometry. The results are displayed in FIGS. 5 and 6, which showthat TCR and CD52 genes have been respectively inactivated in asignificant proportion of the analyzed cells.

Example 3: Functional Analysis of the Engineered T-Cells Electroporatedwith a Monocistronic mRNA Encoding for an Anti-CD19 Single ChainChimeric Antigen Receptor (CAR)

To verify that genome engineering did not affect the ability of theengineered T-cells to present anti-tumor activity when provided with achimeric antigen receptor (CAR CD19), The T-cells previously targetedusing Cas9 and the specific guide RNA targeting TCRa, were furthertransfected with 10 μg of RNA encoding an anti-CD19 CAR. 24 hours later,T cells were incubated for 4 hours with CD19 expressing Daudi cells. Thecell surface upregulation of CD107a, a marker of cytotoxic granulerelease by T lymphocytes (called degranulation) was measured by flowcytometry analysis (Betts, Brenchley et al. 2003).

5×10⁶ T cells preactivated several days (3-5) with anti-CD3/CD28 coatedbeads and IL2 were resuspended in cytoporation buffer T, andelectroporated in 0.4 cm cuvettes without mRNA or with 10 μg of mRNAencoding a single chain CAR

24 hours post electroporation, cells were stained with a fixableviability dye eFluor-780 and a PE-conjugated goat anti mouse IgG F(ab′)2fragment specific to assess the cell surface expression of the CAR onthe live cells. The data is shown in the FIG. 20. A indicates that thevast majority of the live T cells electroporated with the monocitronicmRNA described previously express the CAR at their surface. 24 hourspost electroporation, T cells were cocultured with Daudi (CD19) cellsfor 6 hours and analyzed by flow cytometry to detect the expression ofthe degranulation marker CD107a at their surface (Betts, Brenchley etal. 2003).

The results showed that TCRa-negative cells and TCRa-positive T-cellshad the same ability to degranulate in response to PMA/ionomycin(positive control) or CD19+ Daudi cells. CD107 upregulation is dependenton the presence of a CD19+. These data suggest that genome engineeringusing Cas9 had no negative impact on the ability of T cells to mount acontrolled anti-tumor response.

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1-52. (canceled)
 53. A method of preparing and administering T-cells forcancer immunotherapy comprising the steps of: (a) providing primaryhuman T-cells from a donor, (b) genetically modifying the primary humanT-cells to eliminate expression of two genes, comprising expressing inthe cells (i) a Cas9 endonuclease fused to a nuclear localization signal(NLS), and (ii) a guide RNA that directs said endonuclease to at leastone targeted locus in a first gene in the T-cell genome, and (iii) aguide RNA that directs said endonuclease to at least one targeted locusin a second gene in the T-cell genome, (c) expanding the geneticallymodified T-cells, and (d) administering at least 10⁴ of the expandedgenetically modified T-cells to a cancer patient.
 54. The method ofclaim 53, comprising administering at least 10⁵ of the expandedgenetically modified T-cells to the cancer patient.
 55. The method ofclaim 53, comprising administering at least 10⁶ of the expandedgenetically modified T-cells to the cancer patient.
 56. The method ofclaim 53, comprising administering at least 10⁷ of the expandedgenetically modified T-cells to the cancer patient.
 57. The method ofclaim 53, comprising administering at least 10⁸ of the expandedgenetically modified T-cells to the cancer patient.
 58. The method ofclaim 53, comprising administering at least 10⁹ of the expandedgenetically modified T-cells to the cancer patient.
 59. The method ofclaim 53, wherein the first gene is the TCRa gene.
 60. The method ofclaim 53, wherein the first gene is the TCRβ gene.
 61. The method ofclaim 53, wherein the second gene is the CD52 gene.
 62. The method ofclaim 53, wherein the second gene is the PD1 gene.
 63. The method ofclaim 53, wherein the second gene is the CTLA4 gene.
 64. The method ofclaim 53, wherein the second gene is the LAG3 gene.
 65. The method ofclaim 59, wherein the second gene is the CD52 gene.
 66. The method ofclaim 59, wherein the second gene is the PD1 gene.
 67. The method ofclaim 59, wherein the second gene is the CTLA4 gene.
 68. The method ofclaim 59, wherein the second gene is the LAG3 gene.
 69. The method ofclaim 60, wherein the second gene is the CD52 gene.
 70. The method ofclaim 60, wherein the second gene is the PD1 gene.
 71. The method ofclaim 60, wherein the second gene is the CTLA4 gene.
 72. The method ofclaim 60, wherein the second gene is the LAG3 gene.